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J. Biol. Chem., Vol. 283, Issue 27, 18582-18590, July 4, 2008

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Hydrogen Peroxide Prolongs Nuclear Localization of NF-B in Activated Cells by Suppressing Negative Regulatory Mechanisms^*

Karine Enesa, Kazuhiro Ito, Le A. Luong, Ingvild Thorbjornsen, Chee Phua, Yasuo To, Jonathan Dean^¶, Dorian O. Haskard, Joseph Boyle, Ian Adcock, and Paul C. Evans¹

From the British Heart Foundation (BHF) Cardiovascular Sciences Unit and Respiratory Sciences, National Heart and Lung Institute, and ^¶the Kennedy Institute of Rheumatology Division, Imperial College London, W12 ONN, United Kingdom

Received for publication, February 19, 2008 , and in revised form, April 28, 2008.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
NF-B transcription factors induce pro-inflammatory molecules (e.g. IL-8) in response to cytokines (e.g. TNF, IL-1β) or other stimuli. In the basal state, they are sequestered in the cytoplasm by inhibitory IB proteins. Pro-inflammatory signaling triggers polyubiquitination of intermediaries (e.g. RIP1), which activate IB kinases that trigger Ser phosphorylation and degradation of IB, thereby promoting nuclear translocation of NF-B. A negative feedback loop exists whereby NF-B drives resynthesis of IB, which promotes export of NF-B from the nucleus to the cytoplasm. This process relies on Cezanne, a deubiquitinating cysteine protease that stabilizes resynthesized IB by removing polyubiquitin from modified intermediaries. H₂O₂ is generated during inflammation. Here we examined the effects of H₂O₂ on NF-B dynamics and pro-inflammatory activation in cultured cells co-stimulated with TNF or IL-1β. Quantitative reverse transcription-PCR and enzyme-linked immunosorbent assay revealed that H₂O₂ enhanced the induction of IL-8 by TNF or IL-1β. We demonstrated by using assays of NF-B nuclear localization and by imaging of live cells expressing a fluorescent form of NF-B that H₂O₂ prolonged NF-B nuclear localization in cells co-stimulated with TNF or IL-1β by suppressing its export from the nucleus. We provide evidence that H₂O₂ suppresses NF-B export by prolonging polyubiquitination of signaling intermediaries, which promotes Ser phosphorylation and destabilization of newly synthesized IB proteins. Finally, we observed that the catalytic activity of Cezanne and its ability to suppress RIP1 polyubiquitination and NF-B transcriptional activity were inhibited by H₂O₂. We conclude that H₂O₂ prolongs NF-B activation in co-stimulated cells by suppressing the negative regulatory functions of Cezanne and IB.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Pro-inflammatory cytokines (e.g. TNF,² IL-1β) drive inflammation by activating the redox-sensitive transcription factor NF-B, which induces pro-inflammatory molecules including chemokines (e.g. IL-8). In unstimulated cells, NF-B is sequestered in the cytoplasm through binding to inhibitory IB molecules, which mask its nuclear localization sequence (1, 2). TNF or IL-1β activate distinct pathways that converge to activate IB kinases (IKK), which phosphorylate IB at Ser residues (2–7). This process is regulated by ubiquitin, a protein that can be covalently attached to Lys residues of other cellular proteins in the form of isopeptide-linked polyubiquitin chains (8). Signaling through TNFR or IL-1/Toll-like receptor relies on modification of RIP1 or TRAF6 signaling intermediaries, respectively, with a "non-classical" form of polyubiquitin linked through Lys-63 that triggers the activation of IKKβ (9–13). In addition, polyubiquitin chains linked through Lys-48 play a well recognized role during NF-B activation by targeting phosphorylated IB proteins for proteasomal degradation, thus liberating NF-B for nuclear entry (2, 14–17).

The resolution of inflammatory responses is regulated by multiple negative feedback mechanisms that act in concert to suppress NF-B activity. A key step in this process is the induction of IB by NF-B in activated cells. Newly synthesized IB proteins translocate to the nucleus, where they target activated NF-B complexes for export to the cytoplasm (18–21). NF-B induces other negative regulators of pro-inflammatory signaling including A20 (22) and its sister molecule Cezanne (23), which belong to the OTU family of deubiquitinating cysteine proteases that can cleave ubiquitin monomers from modified proteins (13, 24–26). Both of these proteins can be recruited to activated TNFRs, where they suppress the activity of pro-inflammatory signaling intermediaries by removing polyubiquitin chains from them (13, 26). Thus suppression of TNFR signaling by A20 and Cezanne stabilizes newly synthesized IB, which inhibits NF-B by removing it from the nucleus.

Reactive oxygen species (ROS) such as hydrogen peroxide are generated in inflamed tissues and may contribute to the pathogenesis of chronic inflammatory diseases including atherosclerosis, rheumatoid arthritis, and chronic obstructive pulmonary disease (27–29). Although numerous studies have identified a role for ROS in regulating signaling to NF-B (30), their potential effects on negative regulators of NF-B have received little attention. Nevertheless, emerging reports suggest that ROS may enhance cellular activation by suppressing the activity of anti-inflammatory enzymes such as histone deacetylases (31) and mitogen-activated protein (MAP) kinase phosphatases (MKPs) (32). Here we demonstrate that H₂O₂ can suppress the negative regulatory functions of IB and Cezanne, thus prolonging NF-B nuclear localization and pro-inflammatory transcriptional responses in activated cells.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Reagents and Antibodies—Human recombinant TNF and IL-1β were obtained commercially (R&D, Minneapolis, MN). Anti-RelA (sc372; Santa Cruz Biotechnology, Santa Cruz, CA), anti-IB (sc-847; Santa Cruz Biotechnology), anti-phosphoIB Ser-32/Ser-36 (Cell Signaling Technology), anti-RIP1 (BD Biosciences Pharmingen, Oxford, UK), anti-HA (Roche Applied Science, Burgess Hill, UK), anti-tubulin (Sigma-Aldrich), and anti-lamin B antibodies (Santa Cruz Biotechnology) were obtained commercially. The generation of anti-Cezanne antibodies has been described (26). 5-(and 6-) chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H₂DCFDA) was obtained from Invitrogen. Other reagents were purchased from Sigma-Aldrich unless otherwise stated.

Plasmids—Expression vectors encoding HA epitope-tagged Cezanne (pHM6-Cezanne) or GFP-tagged Cezanne (pEGFP-Cezanne) have been described (23). An expression vector containing a dsRed-tagged version of RelA (pRelA-dsRed) was described previously (33).

Cell Lines and Transfection—HeLa and A549 cells were cultured using Dulbecco's modified Eagle's medium, 10% fetal-calf serum, supplemented with 100 units/ml penicillin G, 100 µg/ml streptomycin. Transient transfection of cells was achieved using Lipofectamine (Invitrogen) according to the manufacturer's instructions.

Measurement of Intracellular ROS—Intracellular ROS were measured in live cells using CM-H₂DCFDA, a probe that forms a fluorescent adduct (dichlorofluorescein) when oxidized. Cells were incubated with 5 µM CM-H₂DCFDA for 15 min and then washed with phosphate-buffered saline prior to experimentation. Fluorescence was quantified by flow cytometry.

ELISA to Detect IL-8—The levels of IL-8 proteins in cell culture supernatants were quantified using a commercial enzyme-linked immunosorbent assay (ELISA, human IL-8 Duo Set; R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. Absorbance (OD) was measured at 450 nm on a microtiter plate reader. Concentrations were obtained by interpolation from standard curves as described previously (34).

Comparative Real-time PCR—Transcript levels were quantified by comparative real-time PCR using gene-specific primers for IL-8 (sense, 5'-TGCCAAGGAGTGCTAAAG-3'; antisense, 5'-CTCCACAACCCTCTGCAC-3'), IB (sense, 5'-CTGATGTCAACAGAGTTACCTACCAG-3'; antisense, 5'-CGTGAACTCTGACTCTGTGTCATAG-3'), and β-actin (sense, 5'-CTGGAACGGTGAAGGTGACA-3'; antisense, 5'-AAGGGACTTCCTGTAACAATGCA-3') purchased from Sigma-Aldrich. Quantitation of GNB2L1 was carried out using primers obtained from Applied Biosystems (Foster City, CA). Total RNA was extracted and reverse-transcribed as described previously (35). Real-time PCR was carried out using the iCycler system (Bio-Rad) and SYBR green master mix (Bio-Rad) according to the manufacturer's instructions. Reactions were incubated at 95 °C for 3 min before thermal cycling at 95 °C for 10 s and 56 °C for 45 s. Reactions were performed in triplicate. Relative gene expression was calculated by comparing the number of thermal cycles that were necessary to generate threshold amounts of product (CT) as described previously (35). CT was calculated for the genes of interest and for the housekeeping gene β-actin. For each cDNA sample, the CT for β-actin was subtracted from the CT for each gene of interest to give the parameter CT, thus normalizing the initial amount of RNA used. The amount of each target was calculated as 2^–CT, where CT is the difference between the CT of the two cDNA samples to be compared.

Assays of NF-B Intracellular Localization—Intracellular localization of endogenous RelA was assessed by immunostaining of paraformaldehyde-fixed cells using anti-RelA antibodies and Alexa Fluor 568-conjugated secondary antibodies followed by laser-scanning confocal microscopy (LSM 510 META; Zeiss, Oberkochen, Germany) following Ref. 35. Image analysis was performed using Velocity software (Improvision, Coventry, UK) to calculate the ratio of RelA present in the nucleus when compared with the cytoplasm. Alternatively, levels of endogenous RelA were measured in cytosolic or nuclear lysates prepared using the NucBuster kit (Novagen, San Diego, CA) by Western blotting using anti-RelA antibodies, horseradish peroxidase-conjugated secondary antibodies, and chemiluminescent detection.

The intracellular localization of RelA was also assessed in real time using cells cultured on glass coverslips in 60-mm dishes. They were transfected with 0.5 µg of mammalian expression vector containing RelA-dsRed and allowed to recover for 48 h. The localization of RelA-dsRed in live cells was assessed using a laser-scanning confocal microscope with a specialized stage that maintained cultures at 37 °C in a 5% CO₂ environment (LSM 510 META; Zeiss, Oberkochen, Germany). Images were made every 3–5 min following stimulation. Image analysis was performed using Velocity software (Improvision, Coventry, UK) to calculate the proportion of RelA-dsRed in the nucleus at multiple time points following stimulation. The duration of nuclear localization was defined as the time interval at which the proportion of nuclear RelA exceeded 20%.

Assay of NF-B Transcriptional Activity—NF-B transcriptional activity was measured using an NF-B reporter (pGL2) as described previously (26). Cells were co-transfected with pNF-luc and pRL-TK (encoding Renilla luciferase to normalize transfection efficiency) using Lipofectamine and incubated for 16 h. Cells were then treated with TNF (10 ng/ml) for 16 h before measurement of NF-B activity. Firefly and Renilla luciferase activity was assessed using the Dual-Luciferase reporter assay kit (Promega, Madison, WI) and luminescence counter (Topcount microplate scintillation; Packard).

Precipitation of TNF Receptor Complexes—HEK293 cells were treated with biotinylated TNF (200 ng/ml) for varying durations and then lysed using 50 mM Tris (pH 7.6), 137 mM NaCl, 1% Triton X-100, 0.5% Nonidet P-40, 1.5 mM MgCl₂, 1 mM EGTA, 5 mM EDTA, 10% glycerol, 0.1 mM Na₃VO₄, 10 µM N-ethylmaleimide, 20 nM MG132, plus a mixture of protease inhibitors (Roche Applied Science, Basel, Switzerland). Lysates were clarified by low speed centrifugation and precleared using protein-G-Sepharose before precipitation of TNFR complexes using streptavidin-coated beads (Roche Applied Science). Beads were then washed extensively using lysis buffer. Precipitated material or lysates were analyzed by Western blotting using specific primary antibodies, horseradish peroxidase-conjugated secondary antibodies, and chemiluminescent detection.

View larger version (30K): [in this window] [in a new window]   FIGURE 1. H2O2 enhanced induction of IL-8 in response to TNF or IL-1β. A, HeLa cells were stimulated with IL-1β (20 ng/ml) for 4 h either in the absence or in the presence of varying concentrations of H2O2. B and C, HeLa cells were stimulated with IL-1β (20 ng/ml) or TNF (10 ng/ml) for varying times either in the presence or in the absence of 100 µM H2O2. A–C, IL-8 transcript levels were quantified by comparative real-time PCR and were normalized by measuring β-actin (A and B) or GNB2L1 (C) transcript levels. Mean values calculated from triplicate wells were pooled from three independent experiments and are shown with standard deviations. D, HeLa or A549 cells were stimulated with IL-1β (20 ng/ml) for 4 h either in the presence or in the absence of H2O2 (100 µM) or remained untreated as a control. E, A549 cells were pretreated with N-acetyl-cysteine (NAC, 10 mM) for 1 h or remained untreated as indicated. They were then stimulated with TNF (10 ng/ml) for 4 h either in the presence or in the absence of H2O2 or remained untreated as a control. D and E, IL-8 protein levels in cell culture supernatants were quantified by ELISA. Mean values calculated from triplicate wells were pooled from three independent experiments and are shown with standard deviations. F, intracellular ROS were measured in HeLa cells, which were preincubated with a redox-sensitive probe (CM-H2DCFDA) and then treated with IL-1β (20 ng/ml) and/or H2O2 (100 µM) for varying times. Mean fluorescence intensities (MFI) were calculated from triplicate measurements of 104 cells and are shown with standard deviations. G, HeLa cells were stimulated with TNF (10 ng/ml) either in the presence or in the absence of 100 µM H2O2. After 2 h, cultures were co-treated with actinomycin D (Act D) to inhibit transcription, and IL-8 transcripts were quantified at varying times by comparative real-time PCR and were normalized by measuring glyceraldehyde-3-phosphate dehydrogenase transcript levels. Data shown represent the proportion of IL-8 mRNA at each time point when compared with IL-8 mRNA levels at the time of actinomycin D treatment. Mean values calculated from triplicate wells were pooled from three independent experiments and are shown with standard deviations.

Statistics—Differences between samples were analyzed using a paired Student's t test and analysis of variance (*, p < 0.05, **, p < 0.01, ***, p < 0.001, as shown in the figure legends).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
H₂O₂ Enhanced Transcriptional Activation of Pro-inflammatory Genes in Response to TNF or IL-1β—We examined whether H₂O₂ could regulate pro-inflammatory activation of HeLa or A549 cells, either alone or in combination with IL-1β or TNF. We observed by comparative real-time PCR that the application of H₂O₂ alone (6–100 µM) had little or no effect on IL-8 transcript levels in HeLa cells (Fig. 1A, compare lane 1 with lanes 6–8). However, H₂O₂ significantly enhanced the production of IL-8 transcripts in a concentration-dependent manner in cells that were co-treated with IL-1β (Fig. 1A, compare lane 2 with lanes 3–5) without affecting cell proliferation or survival (data not shown). Thus we used H₂O₂ at 100 µM in subsequent experiments. In time course experiments, we observed that co-stimulation with H₂O₂ significantly enhanced the induction of IL-8 transcripts by IL-1β or TNF at both early and late time points (Fig. 1B, compare lanes 4, 6, 8, and 10 with lanes 5, 7, 9, and 11; Fig. 1C, compare lanes 3 and 5 with lanes 4 and 6). Similarly, we observed by ELISA of cell culture supernatants that the application of H₂O₂ significantly enhanced the production of IL-8 protein in cells that were co-treated with IL-1β (Fig. 1D, compare lanes 3 and 4 with lanes 7 and 8) or TNF (Fig. 1E, compare lanes 5 and 7).

View larger version (76K): [in this window] [in a new window]   FIGURE 2. H2O2 prolongs nuclear localization and enhances DNA binding of NF-B in activated cells. HeLa cells were stimulated with IL-1β (20 ng/ml) or TNF (10 ng/ml) for varying times either in the presence or in the absence of H2O2 (100 µM) or remained untreated as a control. A, nuclear lysates were tested by Western blotting using anti-RelA antibodies or by using anti-lamin B antibodies to normalize protein levels. RelA protein levels were quantified by densitometry and normalized by measuring lamin B protein levels. Mean values calculated from triplicate measurements are shown with standard deviations. B, intracellular localization of RelA was assessed by immunostaining followed by confocal microscopy. Representative images are shown (upper panels). Mean quantities of nuclear RelA were pooled from three independent experiments and are shown with standard deviations (lower panel).

The potential effects of H₂O₂ on IL-8 mRNA stability were assessed using actinomycin D to block de novo transcription in activated cells. We observed that H₂O₂ did not alter the rate of decay of IL-8 transcript levels in cells co-treated with TNF (Fig. 1G). Taken together, these observations suggest that H₂O₂ can enhance transcriptional activation of IL-8 in response to IL-1β or TNF by altering intracellular redox.

H₂O₂ Prolongs Nuclear Localization and Enhances DNA Binding of NF-B in Activated Cells—We examined whether H₂O₂ modulated NF-B activation by IL-1β or TNF, which is a central event in IL-8 induction. Western blotting or immunostaining revealed that RelA accumulated in the nucleus in response to 15–30 min of treatment with IL-1β (Fig. 2A, upper panel, and Fig. 2B, compare lanes 1 and 2) or TNF (Fig. 2A, compare lanes 6 and 7) but subsequently localized to the cytoplasm following 120 min of treatment (Fig. 2A, compare lanes 2 and 3 with lanes 7 and 8; Fig. 2B, compare lanes 2 and 3). Co-stimulation with H₂O₂ and IL-1β or with H₂O₂ and TNF led to increased nuclear localization of NF-B at late (120–240 min) time points (Fig. 2A, compare lanes 3 and 5 and lanes 8 and 10; Fig. 2B, compare lanes 3 and 5) without significantly affecting nuclear RelA expression at early time points (30 min) (Fig. 2A, compare lanes 2 and 4 and lanes 7 and 9; Fig. 2B, compare lanes 2 and 4). Thus we conclude that co-stimulation with H₂O₂ significantly enhanced nuclear localization of NF-B and binding to DNA sequences in cells that were co-stimulated with IL-1β or TNF for 120–240 min. These findings were supported by in vitro assays, which revealed that H₂O₂ altered the kinetics of NF-B activation in response to IL-1β by enhancing DNA binding following 120 or 240 min of co-treatment (data not shown).

View larger version (23K): [in this window] [in a new window]   FIGURE 3. H2O2 suppresses export of RelA from the nucleus in IL-1β-treated cells. HeLa cells expressing pRelA-dsRed were either treated with IL-1β (20 ng/ml) or co-treated with IL-1β (20 ng/ml) and H2O2 (100 or 200 µM). The intracellular localization of RelA-dsRed was then assessed every 3–5 min by confocal microscopy. A, representative images are shown from single cells. B, image analysis was used to calculate the proportion of RelA-dsRed in the nucleus at multiple time points following stimulation. Mean values calculated from multiple cells were analyzed by non-linear regression to generate curves-of-best-fit, which are shown. C, the time taken for nuclear pRelA-dsRed to reach maximal levels was defined for multiple cells. Mean times of peak nuclear fluorescence are shown with standard deviations (left panel). The duration of nuclear localization was defined for multiple cells. Mean durations of nuclear RelA-dsRed were calculated from multiple cells and are shown with standard deviations (right panel).

View larger version (39K): [in this window] [in a new window]   FIGURE 4. H2O2 promotes Ser-32/Ser-36 phosphorylation and degradation of newly synthesized IB proteins. A and B, HeLa cells were stimulated with IL-1β (20 ng/ml) or TNF (10 ng/ml) for varying times either in the presence or in the absence of 100 µM H2O2 or remained untreated as a control. A, cytosolic lysates were tested by Western blotting using anti-IB antibodies or by using anti-tubulin antibodies to normalize protein levels (upper panels). IB levels were quantified by densitometry and normalized by measuring tubulin protein levels. Mean values calculated from triplicate measurements were pooled from two experiments and are shown with standard deviations (lower panels). B, IB transcript levels were quantified by comparative real-time PCR and were normalized by measuring β-actin transcript levels. Mean values calculated from triplicate wells were pooled from three experiments and are shown with standard deviations. C, the effect of H2O2 on Ser-32/Ser-36 phosphorylation of newly synthesized IB was assessed. HeLa cells were stimulated with IL-1β (20 ng/ml) for varying times either in the presence or in the absence of H2O2 or remained untreated as a control. Cytosolic lysates were tested by Western blotting using anti-phosphoIB (pIB) Ser-32/Ser-36 or anti-IB antibodies or by using anti-tubulin antibodies to normalize protein levels (left panels). Levels of phosphorylated IB were quantified by densitometry and normalized by measuring total IB levels (right panel).

View larger version (37K): [in this window] [in a new window]   FIGURE 5. H2O2 prolongs TNFR signaling by inhibiting an anti-inflammatory deubiquitinating enzyme. A, RIP1 ubiquitination was measured by precipitation of TNF·TNFR complexes from HeLa cells using biotinylated TNF (200 ng/ml). Cells were transfected with an expression vector encoding Cezanne (pHM6-Cez) or remained untransfected. Cultures were subsequently stimulated with biotinylated TNF for varying times either in the presence or in the absence of 100 µM H2O2 before preparation of cytosolic lysates. Alternatively, cytosolic lysates were made from untreated cells as a control and then supplemented with biotinylated TNF. Streptavidin-coated beads were then used to precipitate TNF·TNFR complexes, which were tested by Western blotting using anti-RIP1 antibodies (upper panel). Cytosolic lysates were tested by Western blotting using anti-RIP1 or anti-Cezanne antibodies (center panels). Images shown are representative of those from three closely similar experiments. Levels of polyubiquitinated RIP1 (polyub.-RIP) were quantified by densitometry. Mean values calculated from triplicate measurements are shown with standard deviations (lower panel). monoub.-RIP, monoubiquitinated RIP1. B, reporter gene assays were performed to assess the effects of H2O2 on NF-B suppression by Cezanne. HeLa cells were co-transfected with pHM6-Cezanne (or empty pHM6), pGL2 (NF-B reporter), and pRL-TK (Renilla luciferase control). After 16 h, cells were stimulated with TNF (10 ng/ml) for 16 h either in the presence or in the absence of 100 µM H2O2. Cell lysates were analyzed, and the ratio of firefly/Renilla luciferase activity was calculated, which is a measure of NF-B activity normalized for transfection efficiencies. Mean values of NF-B activity calculated from triplicate wells were pooled from three experiments and are shown with standard deviations. C and D, a thiol-reactive probe (HAUbVME) was used to assess the effects of H2O2 on the catalytic activity of Cezanne. C, HeLa cells expressing GFP-Cezanne were treated with TNF (10 ng/ml) either in the presence or in the absence of 100 µM H2O2 or remained untreated as a control. Cytosolic lysates were incubated with HAUbVME probe or were incubated in the absence of probe as a control. Cezanne linked covalently to probe was detected by Western blotting using anti-HA epitope antibodies (lower panel). Levels of Cezanne in cytosolic lysates were normalized by Western blotting using anti-Cezanne antibodies (anti-Cez., lower panel). Results are representative of two independent experiments. D, cytosolic lysates made from cells expressing GFP-tagged Cezanne were incubated with HAUbVME probe in the presence of varying concentrations of H2O2. Alternatively, lysates were incubated with probe in the presence of a reducing agent (dithiothreitol (DTT)). Cezanne linked covalently to probe was detected by Western blotting using anti-HA epitope antibodies.

Comparative real-time PCR revealed that co-treatment with H₂O₂ enhanced the induction of IB transcripts by IL-1β or TNF (Fig. 4B). We conclude therefore that H₂O₂ suppresses newly synthesized IB at a post-transcriptional level. We next examined whether H₂O₂ regulates phosphorylation of resynthesized IB proteins at Ser-32/Ser-36, a modification that is known to target IB for proteasomal degradation. We observed that serine phosphorylation of newly synthesized IB was significantly greater in cells co-treated for 1 h with H₂O₂ and IL-1β when compared with cells treated with IL-1β alone (Fig. 4C, compare lanes 5 and 7). Thus we conclude that H₂O₂ suppresses export of RelA from the nucleus in activated cells by promoting Ser phosphorylation and destabilization of newly synthesized IB proteins.

H₂O₂ Prolongs TNFR Signaling by Inhibiting an Anti-inflammatory Deubiquitinating Enzyme—We examined whether phosphorylation and destabilization of newly synthesized IB by H₂O₂ in activated cells was associated with prolonged activity of upstream signaling intermediaries. The effects of H₂O₂ on the recruitment of RIP1 to the TNFR and subsequent polyubiquitination were assessed following purification of TNFR complexes using biotinylated TNF. Western blotting revealed that TNF induced transient polyubiquitination of RIP1 in TNFR complexes, which was observed following 2 or 5 min of stimulation but had declined by 20 min (Fig. 5A, compare lanes 1, 2, 6, and 10). Co-treatment of cells with H₂O₂ altered the kinetics of RIP1 polyubiquitination in response to TNF by significantly reducing RIP1 modification following 2 or 5 min of co-treatment (compare lanes 2 and 3 with lanes 6 and 7) and significantly enhancing it at 20 min (compare lanes 10 and 11). Thus although TNFR signaling to NF-B was initially retarded, it was significantly prolonged in cells that were co-treated with H₂O₂.

The export of NF-B from the nucleus to the cytoplasm in chronically stimulated cells relies on the induction of Cezanne, a deubiquitinating cysteine protease that suppresses TNFR signaling to IB by cleaving ubiquitin from modified RIP1 (26). Given the exquisite sensitivity of cysteine proteases to oxidative stress, we reasoned that H₂O₂ may prolong RIP1 polyubiquitination by inhibiting the catalytic activity of Cezanne. Western blotting revealed that overexpression of Cezanne significantly reduced the buildup of polyubiquitinated RIP1 in cells treated with TNF at 5 min but not 20 min (Fig. 5A, compare lanes 6 and 8 with lanes 10 and 12) but had little or no effect in cells that were co-treated with TNF and H₂O₂ at both 5 and 20 min (compare lanes 7 and 9 and lanes 11 and 13). In addition, we observed by reporter gene assay that H₂O₂ significantly reduced the capacity of overexpressed Cezanne to suppress NF-B transcriptional activity in response to TNF (Fig. 5B, compare lanes 3 and 6). We conclude therefore that H₂O₂ suppresses the capacity of Cezanne to inhibit TNFR signaling to NF-B in cultured cells.

We assessed the effects of H₂O₂ on the catalytic activity of Cezanne using an assay employing a ubiquitin-derived thiol-reactive probe (HAUbVME), which is known to bind specifically to the catalytic cysteine of deubiquitinating enzymes (36). Incubation of probe with lysates from cells transfected with GFP-Cezanne revealed a prominent band corresponding to ubiquitin-modified Cezanne (Fig. 5C, compare lanes 1 and 2; Fig. 5D). We confirmed that probe binding observed in these experiments was due to Cezanne sequences by demonstrating that it was not observed using lysates from untransfected cells (data not shown). We observed that probe binding to Cezanne was not influenced by treatment of cells with TNF alone (Fig. 5C, compare lanes 4 and 8 with lane 2) but was reduced by co-treatment of cells with TNF and H₂O₂ (compare lanes 6 and 10 with lane 2). In addition, probe binding to Cezanne was suppressed by the addition of H₂O₂ to reaction mixtures in vitro in a concentration-dependent manner (Fig. 5D, compare lanes 2–5 with lane 6). Taken together, our findings indicate that the catalytic activity of Cezanne can be inhibited by H₂O₂. We conclude that the ability of H₂O₂ to prolong RIP1 polyubiquitination in TNF-treated cells can potentially be explained by its capacity to inhibit the catalytic activity of Cezanne.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Chronically inflamed tissues are characterized by a complex milieu of pro-inflammatory cytokines (e.g. TNF and IL-1β) and ROS (e.g. H₂O₂). A potential role of ROS in the pathogenesis of chronic inflammatory diseases has been suggested from numerous in vivo studies in which inflammatory processes have been suppressed by administration or overexpression of anti-oxidant enzymes (37–41) or have been enhanced by deletion of genes encoding antioxidants (42, 43).

Studies of cultured cells have revealed that ROS have profound effects on numerous physiological activities including pro-inflammatory activation, which can be altered by ROS through poorly defined mechanisms. Numerous reports have indicated that NF-B-dependent transcription is a redox-sensitive process (30). However, the effects of oxidative stress on cellular activation are complex and vary between particular types of cells, ROS, and co-activating stimuli. For example, H₂O₂ has been shown to be an direct inducer of NF-B transcriptional activity and pro-inflammatory activation in some cell types (e.g. T-cells, skeletal muscle myotubes, and human breast MCF-7 cells) but not others (e.g. human microvessel endothelial cells and A549 epithelial cells) (44–46). In addition, H₂O₂ can enhance or prolong cellular activation in response to pro-inflammatory cytokines in several cell types (47, 48).

The molecular mechanisms by which ROS alter signaling to NF-B remain uncertain and are likely to operate at multiple levels. Previous studies have suggested that H₂O₂ stimulates IB phosphorylation at Tyr-42 via Syk protein-tyrosine kinase, which leads to disassociation of NF-B, which can subsequently enter the nucleus (45). Others have shown that H₂O₂ can activate IKK by triggering phosphorylation of serine residues in the activation loop (49), a process that leads to serine phosphorylation and destabilization of IB. In addition, NF-B proteins are themselves sensitive to oxidation, a modification that decreases DNA binding (50).

Here we examined the effects of H₂O₂ on cellular activation in response to TNF or IL-1β. We chose to treat cells with up to 100 µM H₂O₂ because we have previously detected this concentration in supernatants from cultures of activated primary human macrophages.³ We observed that H₂O₂ and pro-inflammatory cytokines (IL-1β, TNF) induced IL-8 in synergy in cultured epithelial cells. We also describe a novel mechanism for the pro-inflammatory effects of H₂O₂, which significantly altered NF-B dynamics in co-stimulated cells by causing it to be retained in the nucleus for prolonged periods. Prior reports suggest that timely NF-B export from the nucleus is essential for suppression of pro-inflammatory processes (18–21). The corollary is that retention of nuclear NF-B in cells exposed to ROS may lead to persistent cellular activation and chronic inflammation.

Activation of NF-B leads to the de novo production of IB, which participates in a negative feedback mechanism by binding nuclear NF-B and exporting it from the nucleus to the cytoplasm (18, 21). Our study suggests that H₂O₂ alters NF-B dynamics in activated cells by triggering Ser-32/Ser-36 phosphorylation and subsequent destabilization of newly synthesized IB proteins. This concept is consistent with previous observations that co-stimulation with H₂O₂ can prolong IKK activation in response to TNF (49). Thus sustained pro-inflammatory signaling in activated cells exposed to H₂O₂ may suppress newly synthesized IB proteins and prevent NF-B export.

To define the level at which H₂O₂ prolongs pro-inflammatory signaling, we assessed its effects on activation of RIP1, which is a key component of the TNFR complex. Recent studies have revealed that TNFR signaling relies on modification of RIP1 with polyubiquitin chains that trigger the activation of IKKβ and TAK1 (11, 13). We demonstrate for the first time that the kinetics of RIP1 polyubiquitination at the TNFR in response to TNF treatment was altered by co-treatment with H₂O₂. Analysis of precipitated TNFR complexes revealed that modification of RIP1 was initially delayed by co-treatment with H₂O₂. The underlying molecular mechanism is uncertain, but we speculate that it could be due to the inhibition by H₂O₂ of ubiquitin-activating enzymes that are known to rely on redox-sensitive Cys residues for catalysis (8). However, the physiological relevance of delayed pro-inflammatory signaling in cells co-treated with H₂O₂ remains uncertain as we did not detect a delay in subsequent transcriptional activity (Fig. 1B). Importantly, we observed that RIP1 polyubiquitination in response to TNF was significantly prolonged in cells that were co-treated with H₂O₂. Thus it is likely that H₂O₂ prolongs NF-B nuclear localization by prolonging TNFR signaling in co-treated cells, which promotes Ser phosphorylation and degradation of newly synthesized IB.

Our group has recently identified a cysteine protease called Cezanne that functions as a negative regulator of TNFR signaling by cleaving ubiquitin monomers from polyubiquitinated RIP1, thus stabilizing IB for NF-B suppression (23, 24, 26). Thus Cezanne inhibits the induction of IL-8 by TNF by shortening the duration of TNFR signaling (26). Given the sensitivity of cysteine proteases to oxidative stress, we reasoned that H₂O₂ may prolong signaling to NF-B by inhibiting the activity of Cezanne. Consistent with this hypothesis, we observed that the catalytic activity of Cezanne and its ability to suppress RIP1 polyubiquitination and NF-B transcriptional activity were inhibited by H₂O₂. We conclude that the ability of H₂O₂ to prolong TNFR signaling can potentially be explained by its inhibitory effects on Cezanne activity.

In summary, we describe a novel mechanism for the pro-inflammatory effects of H₂O₂, which prolonged nuclear localization of NF-B in activated cells by suppressing the negative regulatory functions of IB and Cezanne. Our novel findings that ROS can inhibit an anti-inflammatory deubiquitinating enzyme are consistent with an emerging theme that oxidative stress can promote chronic inflammation by inhibiting the catalytic activity of anti-inflammatory enzymes (31, 32).

	FOOTNOTES

 
^* This work was supported by grants from the British Heart Foundation, Kidney Research UK, and National Heart and Lung Institute. 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.

¹ To whom correspondence should be addressed: BHF Cardiovascular Sciences Unit, National Heart and Lung Institute, Imperial College London, Hammersmith Campus, Du Cane Road, London W12 ONN, UK. Tel.: 44-20-83831619; Fax: 44-20-83831640; E-mail: paul.evans{at}imperial.ac.uk.

² The abbreviations used are: TNF, tumor necrosis factor; IL, interleukin; ELISA, enzyme-linked immunosorbent assay; IKK, IB kinases; ROS, reactive oxygen species; CM-H₂DCFDA, 5-(and 6-) chloromethyl-2',7'-dichlorodihydrofluorescein diacetate; GFP, green fluorescent protein; dsRed, Discosoma species red fluorescent protein; HA, hemagglutinin; Ub, ubiquitin; VME, vinylmethyl; CT, cycle threshold.

³ J. Boyle, unpublished results.

	ACKNOWLEDGMENTS

 
We thank Prof. Gerald Cohen, University of Leicester, for kindly providing biotinylated TNF and Prof. Michael White, University of Liverpool, for kindly providing pRelA-dsRed plasmid.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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