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Originally published In Press as doi:10.1074/jbc.M706420200 on October 5, 2007

J. Biol. Chem., Vol. 282, Issue 50, 36199-36205, December 14, 2007
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Regulation of Peroxiredoxins by Nitric Oxide in Immunostimulated Macrophages*Formula

Alexandre Diet{ddagger}1, Kahina Abbas{ddagger}1, Cécile Bouton{ddagger}, Blanche Guillon{ddagger}, Flora Tomasello§, Simon Fourquet§, Michel B. Toledano§, and Jean-Claude Drapier{ddagger}2

From the {ddagger}Institut de Chimie des Substances Naturelles CNRS, and §Laboratoire Stress Oxydants et Cancer, Institut de Biologie et de Technologies de Saclay, Commissariat à l'Energie Atomique-Saclay, 91190 Gif-sur-Yvette, France

Received for publication, August 3, 2007 , and in revised form, September 27, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Reactive oxygen species and nitric oxide (NO) are capable of both mediating redox-sensitive signal transduction and eliciting cell injury. The interplay between these messengers is quite complex, and intersection of their signaling pathways as well as regulation of their fluxes requires tight control. In this regard, peroxiredoxins (Prxs), a recently identified family of six thiol peroxidases, are central because they reduce H2O2, organic peroxides, and peroxynitrite. Here we provide evidence that endogenously produced NO participates in protection of murine primary macrophages against oxidative and nitrosative stress by inducing Prx I and VI expression at mRNA and protein levels. We also show that NO prevented the sulfinylation-dependent inactivation of 2-Cys Prxs, a reversible overoxidation that controls H2O2 signaling. In addition, studies using macrophages from sulfiredoxin (Srx)-deficient mice indicated that regeneration of 2-Cys Prxs to the active form was dependent on Srx. Last, we show that NO increased Srx expression and hastened Srx-dependent recovery of 2-Cys Prxs. We therefore propose that modulation by NO of Prx expression and redox state, as well as up-regulation of Srx expression, constitutes a novel pathway that contributes to antioxidant response and control of H2O2-mediated signal transduction in mammals.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Macrophages participate in many important functions, including phagocytosis, iron recycling, and host defense, and produce the autacoid-like reactive oxygen species (ROS)3 and nitric oxide (NO) in response to inflammatory cytokines and bacterial products. It has long been reported that ROS and reactive nitrogen species are effectors of an innate immune response (1), but there is increasing evidence that both ROS, and particularly H2O2, and NO also operate as signaling molecules to mediate various responses, including cell growth, angiogenesis, and apoptosis (2, 3). Thus, H2O2 is now recognized as an important intracellular messenger that is physiologically produced by many cells in response to extracellular stimuli like cytokines and growth factors (4). Second messenger functions mediated by H2O2 signaling include activation of mitogen-activated protein kinase (5), modulation of the cell cycle (6, 7), inhibition of tyrosine and lipid phosphatases (8, 9), and protein sumoylation (10). Such signaling pathways imply a tight control of H2O2 production and elimination.

Peroxiredoxins (Prxs) constitute an important peroxidase family that uses the reactivity of the cysteine residues to reduce H2O2 and other peroxides. Reaction of H2O2 with Prxs is fast as indicated by recent reassessment of the kinetic values (11, 12). Further, in addition to their antioxidant function, Prxs have been shown to regulate cell signaling by H2O2 by modulating its fluxes and intracellular levels (13, 14). It is also worth noting that Prxs can reduce peroxynitrite (15, 16). Mammals carry six Prx enzymes that distribute in the three Prx subtypes with four typical 2-Cys Prxs (I-IV), one atypical 2-Cys Prx (Prx V), and one 1-Cys Prx (Prx VI) (17). Typical 2-Cys Prxs have the unique feature of undergoing substrate-mediated inactivation by overoxidation of their catalytic cysteine to a sulfinic acid (R-SO2H). Overoxidation only occurs during enzymatic cycling and is proportional to the amount of substrate under both non-saturating and saturating conditions (15). The fact that inactivation by overoxidation is both unique to eukaryotic Prxs and reversible by ATP-dependent reduction of the Prx Cys-SO2H by sulfiredoxin (Srx or npn3) and sestrins (18–22) had led to the suggestion that it is an acquired gain of function selected for regulating intracellular H2O2 fluxes and signaling (23). Hence, 2-Cys Prx activity is controlled both by the levels of its substrate H2O2 and by the activity of sulfinyl reductases, and this dual control is likely important for regulating H2O2 signaling.

In this report, we have investigated the impact of NO on the expression of Prxs, Srx, and sestrins in murine macrophages. We provide a global view of the expression of the six mammalian Prxs in macrophages that produce NO upon stimulation with interferon {gamma} (IFN-{gamma}) and lipopolysaccharide (LPS). We show that gene expression of Prx I, V, and VI and of Srx was increased in stimulated macrophages. Up-regulation of Prx I and VI, but not Prx V, was mediated by NO. We also report that NO decreases spontaneous and H2O2-induced Prx sulfinylation and hastens recovery upon H2O2-induced Prx sulfinylation, thus pointing to a role for NO in overoxidation prevention and reactivation of 2-Cys Prx.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents—Recombinant mouse IFN-{gamma} (specific activity 2 x 106 units/mg) was from R&D Systems, Abigdon, UK). Escherichia coli LPS, N-(3-(aminomethyl-benzyl-acetamidine)) (1400W), phorbol 12-myristate 13-acetate (PMA), tert-butyl hydroperoxide, and cycloheximide were from Sigma. S-ethylisothiourea and the nitric oxide donor diethylenetriamine NONOate (DETA-NO) were from Cayman Chemical (Ann Arbor, MI). Glucose oxidase was from Calbiochem.

Cell Culture and Treatments—Protocols involving animal experimentation were approved by a national animal care committee. Bone marrow cells were obtained by flushing femurs of WT C57BL/6 mice and of NOS2–/– or Srx–/– mice. Bone marrow-derived macrophages (BMM) were differentiated from bone marrow cells by culture in RPMI 1640 (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen) and 10% L929 cell-conditioned medium. The phenotype of BMM was verified by fluorescence-activated cell sorter. It was shown that >95% of the cells expressed the monocyte/macrophage marker CD11b. BMM were stimulated or not with IFN-{gamma} and/or E. coli LPS at the concentrations and for the times indicated in the figure legends. When indicated, BMM were treated with the nitric oxide donor DETA-NO. Its decomposition rate was determined by the loss of the chromophore at 252 nm.

Preparation of Cell Extracts—BMM cells were washed two times with cold phosphate-buffered saline and lysed in 0.5% Triton X-100 in 100 mM Tris, pH 7.4, containing protease inhibitor mixture Set III (Calbiochem). Cell lysate was then centrifuged at 10,000 x g at 4 °C for 10 min, and the protein content of supernatant was determined spectrophotometrically at 595 nm by using the Bio-Rad protein assay.

Antibodies and Immunoblot Analysis—Anti-Prx I antibody was from Upstate/Chemicon, anti-Prx III antibody was from Abcam, and antibodies to Prx II, Prx VI, Prx (I-IV)-SO2H, and Prx VI-SO2H were from LabFrontier (Seoul, South Korea). The anti-Srx antibody was a purified rabbit polyclonal serum prepared by Neosystem (Strasbourg, France). Anti-vinculin antibody was from Sigma-Aldrich. Cell lysates were fractionated by SDS-PAGE in 12% (Prx) or 15% (Srx) polyacrylamide gel under reducing conditions. After the electrophoretic run and protein immobilization, nitrocellulose membranes (Amersham Biosciences-GE Healthcare) were blocked with Tris-Tween-buffered saline containing 5% nonfat milk and incubated with primary antibodies. Proteins were visualized with horseradish peroxidase secondary antibody (Dako) using enhanced chemiluminescence assay (Millipore) or fluorescent secondary antibodies coupled to either LI-COR IRDyeTM 700 or IRDyeTM 800.

RNA Extraction and Real-time Quantitative PCR—Total RNA was extracted from BMM cells using the SV Total RNA Isolation System (Promega) according to the manufacturer's protocol. Transcription of total RNA was performed using the Moloney murine leukemia virus reverse transcriptase (Promega) and random primers. Quantitative real-time PCR was performed using a Light Cycler, and the detection of amplification products was carried out using the Light Cycler-DNA Master SYBR Green I kit (Roche Diagnostics). The generation of specific PCR products was confirmed by melting curve analysis. Data were analyzed with Light Cycler 3.5 software. Quantification was performed relative to the 18 S rRNA. All assays were performed in triplicate.

Nitrite Measurement—Nitrite, the stable end product of NO, was quantified in culture medium by using the Griess reagent. Briefly, 200 µl of medium were reacted with 800 µl of Griess reagent (0.5% sulfanilamide and 0.05% N-(1-naphthyl)ethylenediamine in 45% acetic acid), and the absorbance was measured at 543 nm. The nitrite concentration was determined from a sodium nitrite standard curve.

H2O2 Generation—Generation of H2O2 was assessed using the fluorescence indicator 2',7'-di-chlorodihydrofluorescein diacetate (H2DCFDA; Molecular Probes, Inc.). BMM were incubated in a 24-well microplate and loaded for 30 min with 20 µM H2DCFDA in Hanks'-buffered solution. They were then stimulated for 30, 60, and 180 min with 500 nM PMA. Dichlorofluoroscein fluorescence was measured with a Victor3 fluorescence microplate reader (PerkinElmer) by emission at 520 nm (excitation, 485 nm).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
NO Increases Prx I and Prx VI Expression in Macrophages—BMM from WT or NOS2–/– mice were stimulated with IFN-{gamma} and LPS. After 18 h, nitrite that had accumulated in the culture medium was quantified, and the expression of the six Prxs was analyzed by quantitative RT-PCR. In stimulated WT BMM, Prx I, Prx V, and Prx VI mRNA expression was noticeably increased compared with unstimulated cells, whereas Prx IV was significantly reduced (Fig. 1A). In contrast, in stimulated NOS2–/– BMM, mRNA level of Prx I, Prx IV, and Prx VI remained unchanged compared with stimulated WT cells, showing that regulation was dependent on NOS2. Still, Prx V mRNA levels remained increased in NOS2–/– BMM, indicating that the up-regulation of this gene by IFN-{gamma} and LPS is independent of NOS2. Prx II and Prx III mRNA levels were not significantly altered by stimulation in either mouse strain. We also used DETA-NO, an NO donor with a long half-life that releases NO at nanomolar concentrations, in the range produced by IFN-{gamma} and LPS-activated macrophages (24). DETA-NO treatment (500 µM) of resting macrophages during 18 h reproduced the effects of endogenous NO produced by BMM on Prx gene expression. Shorter exposures to DETA-NO revealed that Prx VI and Prx I mRNA levels increased 2- and 3-fold after 4 and 8 h, respectively (supplemental Fig. S1), suggesting that regulation is at least in part transcriptional. Moreover, the use of DETA-NO that had been left to decompose for 7 days at 37 °C (>8 half-lives) had no effect on gene expression, indicating that neither DETA nor nitrite is responsible for the regulation of Prxs observed with DETA-NO (not shown). Altogether, these data point to a physiological role of NO in the selective regulation of Prx I, IV, and VI mRNA levels.

We further checked whether the NO-dependent increase in Prx I and VI transcript levels also occurred at protein level. Higher Prx I and VI protein levels were seen in BMM stimulated with IFN-{gamma} and LPS, but not when cells were stimulated in the presence of NOS inhibitors or using cells explanted from NOS –/–2-deficient mice (Fig. 1B). Prx I and Prx VI protein levels were also increased in BMM incubated with DETA-NO, in a dose-dependent manner. In contrast, Prx II and III protein levels were not sensitive to NO (Fig. 1C). On the whole, these results highlight the role of NO in up-regulation of Prx I and VI protein expression.


Figure 1
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  		FIGURE 1.
Effect of NO on Prx expression. A, differential Prx expression between WT and NO2–/– BMM. RNA from BMM left untreated (control), stimulated with 100 units/ml IFN-{gamma} and 500 ng/ml LPS, or exposed to 500 µM DETA-NO for 18 h was tested for Prx gene expression by quantitative RT-PCR. The –fold difference in mRNA expression, normalized to 18 S ribosomal protein, is indicated. Error bars, mean ± S.D. from at least three independent experiments. B and C, expression of Prx protein was measured by immunoblotting in BMM (WT or NOS2–/– when indicated). BMM were left untreated (Ctrl), stimulated with 100 units/ml IFN-{gamma} and 500 ng/ml LPS (I/L) in the presence or not of 25 µM 1400W and 100 µM S-ethylisothiourea, two NOS inhibitors (In), or exposed to 50 or 500 µM DETA-NO (D50 or D500). After 20 h, accumulation of nitrite was used to determine NOS2-derived NO production, and cell lysates (20 µg of protein) were analyzed by immunoblotting with the indicated antibodies. Representative experiments are shown of at least three performed (nd, not detectable).

 
Effect of NO on Prx Overoxidation—The active site cysteine of eukaryotic Prxs undergoes substrate-mediated oxidation to the sulfinic acid form (SO2H), which inactivates enzyme activity (25, 26). Based on the notion than NO induces cysteine sulfinylation in vitro (27, 28), we evaluated whether NO would also promote overoxidation of Prxs. Using an antibody that immunoreacts with the sulfinylated form of the four 2-Cys Prxs (I–IV), we thus monitored the level of overoxidized Prxs in macrophages that were stimulated to produce NO (Fig. 2A). In lysates of H2O2-challenged RAW 264.7 macrophages used as a positive control, the anti-Prx-SO2H antibody revealed the presence of two main bands and one barely visible band. Based on molecular mass and published data (19, 29), the lower band is sulfinylated Prx I/II and the upper main band corresponds to sulfinylated Prx III, whereas the uppermost faint band is presumably Prx IV. In control WT BMM, a unique anti-Prx-SO2H immunoreactive band was detected at the size of Prx I/II. This low steady-state oxidation level is expected to be the consequence of ROS basal production by macrophages. Surprisingly, in IFN-{gamma}- and LPS-stimulated macrophages, Prx overoxidation was not or barely detectable, but in NOS2–/–-stimulated macrophages and in macrophages stimulated in the presence of NOS inhibitors, overoxidation was still present at the levels of unstimulated cells. To identify the NO-derived species responsible for the decrease in Prx overoxidation observed in stimulated macrophages, we tested the effect of DETA-NO. Exposure of BMM to DETA-NO also lessened Prx overoxidation (Fig. 2B). This effect was observed at DETA-NO concentrations as low as 50 µM and was markedly increased at 500 µM. Time course experiments indicated that the effect of NO on Prx oxidation occurred early, with an ~50% decrease at 2 h after addition of DETA-NO (Fig. 2B, lower panel).


Figure 2
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  		FIGURE 2.
Endogenous and exogenous NO decrease basal overoxidation of Prxs. WT and NOS2–/– BMM were stimulated with 100 units/ml IFN-{gamma} and 500 ng/ml LPS (I/L) in the presence or not of 25 µM 1400W and 100 µM S-ethylisothiourea, two NOS inhibitors (In)(A), or exposed to 50 or 500µM DETA-NO (B). As a positive control for the detection of Prx overoxidation, lysate of H2O2-challenged RAW 264.7 macrophage cell line was loaded (upper panel, right corner). Based on molecular mass and Refs. 19 and 29, the uppermost (and minor) band is Prx IV, the middle band is Prx III, and the lower (major) band is Prx I/II. Lower panel, WT BMM were exposed to 500µM DETA-NO for increasing times ranging from 1 to 24 h, and cell lysates were assessed for expression of overoxidized Prx. Vinculin was used as a loading control. The experiment shown is representative of at least three performed.

 


Figure 3
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  		FIGURE 3.
Effect of NO on the H2O2-triggered overoxidation of Prxs. A, BMM were treated with 100 units/ml IFN-{gamma} and 500 ng/ml LPS or exposed to 500 µM DETA-NO for 18 h. Cell monolayers were then exhaustively washed and exposed for 30 min to 100 µM H2O2 (A) or to 100 µM tert-butyl-hydroperoxide (t-BOOH) (B) and for 3 h to 100 milliunits of glucose oxidase or to 500 nM PMA (C). 2-Cys Prxs were then assessed for overoxidation by immunoblotting with a specific antibody. Vinculin was used as a loading control. The experiment shown is representative of at least three performed.

 
We next evaluated whether NO would also alter the overoxidation of Prxs induced by exogenous H2O2. BMM were treated with IFN-{gamma} and LPS or were exposed to DETA-NO for 16 h. After extensive washings, they were then challenged with a bolus of H2O2 (Fig. 3A) with the membrane-permeant pro-oxidant agent tert-butyl-hydroperoxide (Fig. 3B) or with H2O2 continuously produced at low concentrations by the glucose/glucose oxidase system (Fig. 3C). Again, H2O2-induced Prx I/II, Prx III, and, to a lesser extent, Prx IV overoxidation was significantly decreased in DETA-NO-treated macrophages as compared with the untreated controls. At least with respect to Prx I/II, similar results were obtained with human epithelial carcinoma (HeLa) cells exposed to exogenous NO (supplemental Fig. S2), pointing to a more general effect on various cell populations and species. We also used PMA, a potent NOX2 activator that stimulates endogenous H2O2 production. Macrophage treatment with PMA indeed led to H2O2 production, as testified by oxidation of the fluorescent probe dichlorofluoroscein diacetate (not shown). PMA also increased the basal levels of sulfinylated 2-Cys Prxs, which was not seen when cells had been previously treated with DETA-NO (Fig. 3C).

We also checked whether 1-Cys Prx VI overoxidation was also affected by NO, using a Prx VI-SO2H-specific antibody. Overoxidized Prx VI was detectable only after exposure of resting BMM to 100 µM H2O2, and prior stimulation of BMM by IFN-{gamma}/LPS or exposure to DETA-NO did not alter Prx VI overoxidation (supplemental Fig. S3). Prx VI protein levels were increased by NO as shown above (Fig. 1B) and after reprobing membranes with an anti-Prx VI antibody (supplemental Fig. S3). Prx V was not evaluated, because this Prx isoform is not sensitive to overoxidation (20).

In summary, these data clearly indicate that NO decreases 2-Cys Prx sulfinylation rather than promoting this form of oxidation. These results could be explained by NO either preventing Prx sulfinylation or favoring its recycling. Based on the simultaneous effect of NO on up-regulation of Prx I expression, it is possible that the resulting increased H2O2-scavenging capacity could act as a factor diminishing Prx I overoxidation. However, the fact that the translation inhibitor cycloheximide did not prevent DETA-NO from abating H2O2-mediated Prx overoxidation (supplemental Fig. S4) indicates that Prx I up-regulation and overoxidation can be dissociated. This result also show that decrease in Prx overoxidation does not require de novo protein synthesis.

Effect of NO on Srx Expression—Sulfinylated 2-Cys Prx (I–IV) can be reduced by two different types of enzymes with ATP-dependent sulfinic acid reductase activity, Srx (18, 21) and the sestrins (22). We therefore investigated the possible involvement of Srx and sestrins in the NO-dependent decline in Prx overoxidation. Sestrin mRNA levels were not significantly modified in NO-producing or DETA-NO-exposed BMM (not shown). In contrast, Srx mRNA levels were significantly up-regulated in IFN-{gamma}/LPS-stimulated macrophages (Fig. 4A), and this increase was dependent on NOS2-derived NO because it was not observed in stimulated macrophages from NOS2–/– mice. A time course experiment showed that the increase in Srx expression in DETA-NO-exposed BMM began as early as 1 h after DETA-NO exposure, peaked after ~5 h, and remained high after 24 h (Fig. 4B). Western blot analyses using an Srx-specific antibody showed that the increase in Srx mRNA levels was paralleled by an increase in Srx protein levels (Fig. 4C).

We sought to determine whether the NO-dependent up-regulation of Srx and the decline in Prx I–IV overoxidation were linked. To this aim, we analyzed the Prx oxidation status in BMM from WT or Srx-deficient mice that had been exposed to DETA-NO and then challenged with 100 µM H2O2. As already shown in Fig. 2, preincubation of WT BMM with DETA-NO decreased both constitutive and H2O2-induced Prx overoxidation (Fig. 5A, compare lane 3 with lane 1 and lane 4 with lane 2). The same results were observed in Srx–/– BMM (compare lane 7 with lane 5 and lane 8 with lane 6), indicating that the effect of NO on the decrease of Prx overoxidation does not involve Srx. To further investigate a possible role of Srx on Prx regeneration, we measured the rate of Prx sulfinic acid reversion after a brief exposure to H2O2. Prx sulfinylation was measured at different time points in BMM preincubated with DETA-NO that were then exposed to H2O2 (100 µM) during 30 min and then washed and re-incubated in fresh medium (Fig. 5B). Densitometric analysis of immunoblots showed that the intensity of the H2O2-induced sulfinic acid signal had decreased by 50% after 1.5 h in WT control BMM and after only 45 min in DETA-NO-treated WT BMM. Furthermore, in lysates of Srx–/– BMM that had been incubated or not with NO, the signal of H2O2-induced sulfinylated Prx did not decrease, remaining high throughout the entire observation period. These results indicate that NO not only prevents Prx sulfinylation but also increases the efficiency of its in vivo reduction. They also confirm the crucial role of Srx in the reduction of sulfinylated 2-Cys Prxs in mammals.


Figure 4
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  		FIGURE 4.
NO enhances Srx expression. A, quantitative RT-PCR was performed on total RNA prepared from WT and NOS2–/– BMM previously stimulated for 20 h with 100 units/ml IFN-{gamma} and 500 ng/ml LPS. Expression of Srx was normalized against the expression of the 18 S ribosomal RNA gene. Mean ± S.D. of three independent experiments. B, BMM were exposed to 500 µM DETA-NO for increasing times, and Srx mRNA expression was measured by quantitative RT-PCR. Relative levels of Srx mRNA are shown as normalized with the 18 S ribosomal RNA gene. Mean ± S.D. of three independent experiments. C, BMM were stimulated with 100 units/ml IFN-{gamma} and 500 ng/ml LPS (I/L) or exposed to 500 µM DETA-NO (D) for 20 h. Cell extracts were tested for Srx expression by an immunoblot analysis with a specific antibody. The experiment shown is representative of at least three performed.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Signaling by oxygen- and nitrogen-derived species is attracting growing attention as its involvement in diverse cellular responses is being disclosed (30, 31). As these mediators are reactive, they can also potentially damage various biomolecules, making their control of prime importance for cell physiopathology (1, 32, 33). Prxs are important cellular peroxide-scavenging enzymes and have been shown to also modulate signaling by H2O. Classical 2-Cys Prxs, which comprise Prx I-Prx IV, undergo reversible hydroperoxide-mediated inactivation by overoxidation of their catalytic cysteine to the sulfinic acid form (16, 23) and reduction by ATP-dependent sulfiredoxin and sestrins (18, 21). This inactivation, being unique to eukaryotic Prxs, has led to suggestions that it is an acquired gain of function selected for regulatory purposes (23). In the present study, we have identified a novel cross-talk between the action of NO and H2O2 in primary macrophages. This cross-talk is based on the effect of NO of decreasing H2O2-induced 2-Cys Prx sulfinylation, of speeding up the regeneration of sulfinylated inactive enzymes, and of up-regulating Prx I, Prx VI, and Srx at mRNA and protein levels. These effects were observed whether NO was endogenously produced by NOS2 or delivered extracellularly by the slow NO donor DETA-NO and when Prxs were sulfinylated by H2O2 that was endogenously produced by stimulated macrophages or applied extracellularly. Our data also provide evidence that Prx V is induced in macrophages stimulated by IFN-{gamma} and LPS, but in contrast to Prx I and Prx VI this induction is independent of NO production. Also, in contrast to what was observed for 2-Cys Prxs, H2O2-mediated overoxidation of 1-Cys Prx VI was not significantly altered by endogenous or exogenous NO, which is consistent with the fact that Srx is not able to reduce sulfinyl-Prx VI (29). Taken together, these results indicate that NO production increases the Prx-dependent antioxidant capacity of macrophages, both by increasing Prx I and VI levels and by preserving typical 2-Cys Prxs in their reduced active form, particularly when macrophages release high amounts of H2O2.


Figure 5
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  		FIGURE 5.
Effect of NO on Prx overoxidation in WT and Srx–/– BMM. A, BMM were left untreated or cultured for 18 h in the presence or not of 500 µM DETA-NO. They were exhaustively washed and challenged by 100 µM H2O2 for 30 min, and the level of Prx-SO2H was assessed by immunoblotting. B, WT and Srx–/– BMM were treated as described above, washed again, and left to recover for the indicated times. Cell extracts were probed for expression of Prx-SO2H by immunoblot analysis. Lower panel, densities of the major band (PrxI/II-SO2H) were normalized to vinculin expression and quantified by scanning densitometry. The dotted line indicates the times corresponding to 50% Prx-SO2H level. The experiment shown is representative of at least three performed.

 


Figure 6
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  		FIGURE 6.
Two-level intersection of NO- and H2O2-signaling pathways. In this proposed scheme, NO produced by immunologically stimulated macrophages first rapidly alleviates overoxidation of 2-Cys Prxs by post-translational modification and then mediates faster recovery to their reduced state via Srx up-regulation.

 
These data are in accordance with the growing body of evidence that NO has antioxidant properties (33–36) and, notably, confers protection against the cytotoxicity of H2O2 (37, 38). More importantly, they provide a mechanistic insight of this protective effect, as depicted in the model of Fig. 6. Prx V is also known as an efficient peroxynitrite reductase (39) and is partly located in mitochondria, a compartment expected to be a site of significant peroxynitrite production (40). Therefore, the increased expression of Prx V in IFN-{gamma}- and LPS-activated macrophages might also represent a protection mechanism for avoiding the long-term toxic effects of NO that is produced by these activated cells. We thus propose that stimulated macrophages are likely to benefit from having a high level of active Prx to limit the collateral negative effects of ROS-mediated and nitrosative stress.

An NO-dependent, but guanylate cyclase-independent, up-regulation of Prx I had been previously observed in LPS-stimulated rat liver macrophages (41), but the mechanism of this regulation was not further documented. The Prx I and Prx VI promoters contain sites for the NO-responsive transcription factor Nrf2 (42–44), and macrophages of Nrf2-deficient mice are unable to induce Prx I expression in response to H2O2 and to electrophilic compounds (45). Similarly, Srx (also referred to as neoplastic progression 3) was highly induced in a strictly Nrf2-dependent manner in the liver of mice fed with the Nrf2-inducer 3H-1,2-dithiole-3-thione, as shown by DNA microarray analysis (46). Moreover, it is well established that NO released from NO donors, including DETA-NO, regulates antioxidant response element-mediated gene expression in mammalian cells (42, 45, 47). It is therefore tempting to propose that NO, via the Nrf2/Keap1 system, contributes to an adaptive response to oxidative and nitrosative stresses by coordinate up-regulation of these three redox enzymes.

The question arises: How does NO affect 2-Cys Prx overoxidation? Our data indicate that NO both prevented catalytic cysteine overoxidation and accelerated the rate of its recovery by sulfiredoxin (18, 21). We found that NO-exposed WT BMM displayed much lower constitutive amounts of Prx-SO2H (see Fig. 2A). As this effect was prominent as early as 2 h after exposing cells to NO (Fig. 2B), it could not be the consequence of faster reduction of the sulfinylated form of Prxs by Srx because induction of the later gene by NO peaked much later, at 5 h (Fig. 4). We also observed that the overoxidation of Prxs that resulted from a 30-min exposure to H2O2 was significantly lower in NO-treated versus untreated macrophages, also implying that the effect could not be a consequence of a more efficient reduction by Srx. In addition, the effect of NO pretreatment on the overoxidation of Prxs by exogenous H2O2 was fully maintained in Srx–/– BMM, further indicating that the effect of NO in preventing 2-Cys Prx overoxidation is unrelated to Srx (Fig. 4). Lipid hydroperoxide metabolites of arachidonic acid, which are commonly produced by stimulated macrophages, have recently been shown to reversibly overoxidize 2-Cys Prxs in cyclooxygenase- or lipoxygenase-overexpressing human cell lines (48). It would be worth considering that NO, by scavenging lipid peroxyl radicals (49, 50), decreases the level of 2-Cys Prx overoxidation. Alternatively, a plausible explanation is that Prxs from NO-producing macrophages are protected from overoxidation by H2O2 by a post-translational modification of its catalytic cysteine residues. Based on the role of S-glutathionylation in the adaptive response to oxidative stress (51, 52), protection of the Prx active site cysteine(s) by NO-induced disulfide formation with glutathione is an appealing hypothesis.

Nevertheless, NO also affected the rate of the Srx-dependent reversion of sulfinylated Prxs. This was deduced from the faster decline in the amount of PrxI/II-SO2H generated by exogenous H2O2 in NO-treated versus untreated macrophages (Fig. 5B).

Our data also revealed that in Srx-deficient BMM, H2O2-induced Prx sulfinylation remained stable over time, indicating that Srx is the major macrophage sulfinyl reductase and that no other enzyme can compensate for its deficiency in these cells. This finding is in keeping with the work of Chang et al. (21) showing that small interfering RNA-mediated Srx silencing delays Prx regeneration in epithelial cells. It also supports the idea that the effect of NO in accelerating Prx regeneration is mediated by NO-induced Srx up-regulation, thus exemplifying the role of Srx in modulating the redox state of Prxs in a physiological setting.

To conclude, by pointing to Prxs as a cross-talk between NO and H2O2 signaling, we propose a novel control mechanism by which physiologically produced NO exerts an antioxidant effect. Increase in the amount of active Prxs is likely to represent a negative feedback loop to protect against excessive stress. It may also contribute to the redox control of the kinase/phosphatase balance. Further, the intrusion of the IFN-{gamma}- and LPS-driven NOS2 as a new player in the Prx/Srx-regulating system of H2O2 flux opens a wide field for investigations of a connection between host defense and cell redox signaling.


   FOOTNOTES
 
* This work was supported by grants from the Agence Nationale de la Recherche (to J.-C. D. and M. B. T.) and from Association pour la Recherche sur le Cancer (to M. B. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S4 and Table S1. Back

1 Both authors contributed equally to this work. Back

2 To whom correspondence should be addressed: ICSN-CNRS, Bât. 27, Ave. de la Terrasse, 91190 Gif-sur-Yvette, France. Fax: 33-1-69-07-72-47; E-mail: drapier{at}icsn.cnrs-gif.fr.

3 The abbreviations used are: ROS, reactive oxygen species; BMM, bone marrow-derived macrophage; NO, nitric oxide; NOS2, nitric-oxide synthase 2; DETA-NO, diethyltriamine-NONOate; PMA, phorbol 12-myristate 13-acetate; Prx, peroxiredoxin; Srx, sulfiredoxin; LPS, lipopolysaccharide; IFN, interferon; WT, wild type; RT-PCR, reverse transcription PCR. Back


   ACKNOWLEDGMENTS
 
We thank Drs. C. H. Cottart (Faculté des Sciences Pharmaceutiques de Paris, France) and J-C. Jeanny (Unité Inserm 598, Paris, France) for kindly providing us with NOS2–/– mice, which were generated by Drs. C. Nathan (Cornell University Department of Medicine) and J. Mudgett (Merck, Rahway, NJ), and Dr. M. Gérard and P. Hery (IBITECS, CEA-Saclay) for the preparation of the Srx–/– mice. We are also thankful to Dr. J. Bignon (ICSN-CNRS) for fluorescence-activated cell sorter analyses and Dr. J. Breton for helpful discussion.



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 EXPERIMENTAL PROCEDURES
 RESULTS
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