Gene expression profiling reveals a signaling role of glutathione in redox regulation Maddalena Fratelli*, Leslie O. Goodwin†, Ulf Andersson Ørom*‡, Sarah Lombardi†, Rossella Tonelli*, Manuela Mengozzi*, and Pietro Ghezzi*§ *Laboratory of Neuroimmunology ‘‘Mario Negri,’’ Institute for Pharmacological Research, 20157 Milan, Italy; and †North Shore–Long Island Jewish Research Institute, Manhasset, NY 11030 Edited by Charles A. Dinarello, University of Colorado Health Sciences Center, Denver, CO, and approved July 22, 2005 (received for review May 27, 2005) Proteins can form reversible mixed disulfides with glutathione reversible oxidations to form disulfides and glutathionylated (GSH). It has been hypothesized that protein glutathionylation may proteins (2–5). The reversibility of protein glutathionylation, represent a mechanism of redox regulation, in a fashion similar to catalyzed by glutaredoxin and, to a lesser extent, thioredoxin (6), that mediated by protein phosphorylation. We investigated makes this posttranslational modification a likely molecular whether GSH has a signaling role in the response of HL60 cells to mechanism by which GSH could act as a redox-dependent hydrogen peroxide (H2O2), in addition to its obvious antioxidant signaling molecule, in analogy with protein phosphorylation. role. We identified early changes in gene expression induced at However, despite the identification of several proteins under- different times by H2O2 treatment, under conditions that increase going glutathionylation (5, 7), in our opinion a signaling role of protein glutathionylation and minimal toxicity. We then investi- glutathionylation is far from being established. gated the effect of prior GSH depletion by buthionine sulfoximine Because glutathionylation is a redox-dependent modification, and diethylmaleate on this response. The analysis revealed 2,016 we reasoned that it might participate in signaling the exposure genes regulated by H2O2. Of these, 215 genes showed GSH- of the cell to oxidants and have undertaken a study to establish dependent expression changes, classifiable into four clusters dis- whether GSH has a signaling role in the response to oxidants. To playing down- or up-regulation by H O , either potentiated or 2 2 this purpose, we performed studies of gene expression profiling inhibited by GSH depletion. The modulation of 20 selected genes in human promyelocytic leukemia HL-60 cells exposed to hy- was validated by real-time RT-PCR. The biological process catego- ries overrepresented in the largest cluster (genes whose up- drogen peroxide (H2O2). Several genes were regulated at dif- regulation was inhibited by GSH depletion) were NF-␬B activation, ferent time points in response to H2O2, under conditions that transcription, and DNA methylation. This cluster also included resulted in increased protein glutathionylation. Using this several cytokine and chemokine ligands and receptors, the redox model, we studied the effect of prior GSH depletion by buthi- regulator thioredoxin interacting protein, and the histone deacety- onine sulfoximine (BSO) (8) and the GSH depletor diethylmal- lase sirtuin. The cluster of genes whose up-regulation was poten- eate (DEM) (9), to investigate, using two-way ANOVA on tiated by GSH depletion included two HSPs (HSP40 and HSP70) and microarray data, whether there are patterns of gene regulation the AP-1 transcription factor components Fos and FosB. This work that require GSH, because these would be the changes where demonstrates that GSH, in addition to its antioxidant and protec- protein glutathionylation might have a role. tive function against oxidative stress, has a specific signaling role in redox regulation. Methods HL60 Cells and GSH Depletion and Treatments. HL60 cells were chemokines ͉ cytokines ͉ hydrogen peroxide ͉ oxidative stress cultured in RPMI medium 1640 with 2 mM L-glutamine͞100 units/ml penicillin͞100 ␮g/ml streptomycin sulfate (all from lutathione (GSH) protects the cell from damage induced by Sigma)͞10% heat-inactivated FCS. Cells were maintained at a Ghigh levels of reactive oxygen species (ROS), defined as concentration between 0.3 ϫ 106 per ml and 1.5 ϫ 106 per ml. oxidative stress (1). Its main antioxidant activity consists of the For GSH depletion, they were incubated at the density of 0.75 ϫ detoxification of peroxides, partly with the aid of various GSH 106 per ml in complete medium with 200 ␮M BSO for 23 h, then peroxidases. Its role as a major thiol antioxidant is demonstrated with 1 mM DEM for 1 additional hour. Twenty-four hours after by the fact that various conditions of oxidative stress are starting the depletion treatment, cell density was 0.75 ϫ 106 as exacerbated by GSH depletion, for instance by inhibitors of its compared to 1.0 ϫ 106 per ml in parallel controls and viability synthesis, and ameliorated by the addition of GSH or its pre- (as determined by Trypan blue exclusion) was Ͼ98% in both cursors, including N-acetylcysteine (NAC). Experimentally, conditions. Control or depleted cells were then incubated with thiol antioxidants, including GSH and NAC, have been used as 50 ␮MH2O2 for 0, 0.5, 2, or 24 h, always in complete medium. tools to investigate the role of ROS in biological systems. In addition to scavenging ROS, GSH can form mixed disul- GSH Determinations. Cells (30 ϫ 106) were suspended in 0.3 ml of fides with proteins, a phenomenon known as protein glutathio- 5% sulfosalycylic acid. GSH was measured both in the super- nylation (2–4). This can occur by various reactions, either by natant and in the fraction released from the washed pellet by ͞ thiol disulfide exchange between protein cysteines and oxidized alkali treatment (protein-bound GSH) (10) using an enzymatic GSH (GSH disulfide, GSSG), by direct oxidation, or through the method (11). NO-mediated formation of S-nitrosothiols (3, 4). The effect of protein glutathionylation is generally considered deleterious in the framework of oxidative stress, because it is one This paper was submitted directly (Track II) to the PNAS office. of the many forms of thiol oxidation induced by ROS. However, Abbreviations: BSO, buthionine sulfoximine; DEM, diethylmaleate; GSH, glutathione; according to the more recent concept of redox regulation, GSSG, GSH disulfide; H2O2, hydrogen peroxide; NAC, N-acetylcysteine; ROS, reactive oxy- several protein cysteines can be defined as ‘‘redox sensitive.’’ gen species; HSP, heat-shock protein. Although disulfide bonds are virtually absent in cytosolic pro- ‡Present address: Biotech Research and Innovation Centre, 2100 Copenhagen, Denmark. teins, due to the highly reducing intracellular environment, §To whom correspondence should be addressed. E-mail: [email protected]. proteomic studies have shown that many of them can undergo © 2005 by The National Academy of Sciences of the USA 13998–14003 ͉ PNAS ͉ September 27, 2005 ͉ vol. 102 ͉ no. 39 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0504398102 Downloaded by guest on September 24, 2021 RNA Isolation and Microarray Hybridization. All experiments were performed in triplicate. Each sample (20 ϫ 106 cells) was lysed in 3 ml of TRIzol Reagent (Life Technologies, Gaithersburg, MD) and RNA extracted according to the manufacturer’s pro- tocol. The obtained RNA was further purified by using the RNeasy (Qiagen, Valencia, CA) system and protocol. Only the samples with the highest purity and integrity (as judged by A260͞280 ratio, on 1% agarose͞formaldehyde gels and on a Bioanalyzer) (Agilent Technologies, Palo Alto, CA) were pooled and further processed. Biotin labeling of total RNA, hybridiza- tion, washing, and scanning of the Affymetrix (Santa Clara, CA) GeneChip Human Genome Hg-U133A were carried out as recommended by the manufacturer. Because there was Ͻ2% variability between replicate gene chips carried out with these procedures, technical replicates were not performed. Biological triplicates (samples from three completely independent exper- iments) were analyzed. Microarray Data Analysis. Image quantification, background sub- traction, and scaling were made with MICROARRAY SUITE (MAS) 5.0 software (Affymetrix) with the default parameters for the statistical algorithm. No further background correction of the data was applied. For signal-dependent normalization, the Qspline method (12) was applied with default settings from the affy-package from Bioconductor (13), using the R language and environment. For statistical analysis of differentially ex- pressed genes, we applied ANOVA in the R environment (14), either one way for the extended time course in control cells (0, 0.5, 2, and 24 h of H2O2 treatment) or two-way for the compa- rable parts of the experiment (control and depleted cells, 0, 0.5, Fig. 1. Experimental design and data analysis diagram. and 2 h of H2O2 treatment), giving values for the significance of differences between the groups (depletion effect), in the groups (H O effect), or with interaction. This gave us a table of four 2 2 Results P values for each gene. Because the distribution of the P values for the interaction in two-way ANOVA was homogeneous, we Experimental Design. HL60 cells were depleted of GSH by over- did not take into account this parameter. Two sets of genes night treatment with 200 ␮M BSO followed by 1-h incubation (affected by H2O2 treatment and not by GSH depletion and with 1 mM DEM. This led to a reduction of 80% in total cellular affected by both conditions) were selected and classified accord- GSH, without affecting cell viability. Cells were then treated ␮ ing to the similarity in their