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Hydrogen Peroxide Induces Murine Macrophage Chemokine Gene Transcription Via Extracellular Signal-Regulated Kinase- and Cyclic Adenosine 5'-Monophosphate (cAMP)-Dependent Pathways: Involvement of NF-B, Activator Protein 1, and cAMP Response Element Binding Protein¹

Centre de Recherche en Infectiologie, Centre Hospitalier Universitaire de Québec, Pavillon du Centre Hospitalier de l’Université Laval, and Département de Biologie Médicale, Faculté de Médecine, Université Laval, Ste-Foy, Québec, Canada

	Abstract

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Hydrogen peroxide (H₂O₂) has been shown to act as a second messenger that activates chemokine expression. In the present study, we investigated the mechanisms underlying this cellular regulation in the murine macrophage cell line B10R. We report that H₂O₂ increases mRNA expression of various chemokines, macrophage-inflammatory protein (MIP)-1/CC chemokine ligand (CCL)3, MIP-1/CCL4, MIP-2/CXC chemokine ligand 2, and monocyte chemoattractant protein-1/CCL2, by activating the extracellular signal-regulated kinase (ERK) pathway and the nuclear translocation of the transcription factors NF-B, AP-1, and CREB. Blockage of the ERK pathway with specific inhibitors against mitogen-activated protein kinase kinase 1/2 and ERK1/ERK2 completely abolished both the H₂O₂-mediated chemokine up-regulation and the activation of all NF studied. Similarly, selective inhibition of cAMP and NF-B strongly down-regulated the induction of all chemokine transcripts as well as CREB and NF-B activation, respectively. Of interest, we detected a significant decrease of NF-B, AP-1, and CREB DNA binding activities by reciprocal competition for these binding sites when either specific cold oligonucleotides (NF-B, AP-1, and CREB) or Abs against various transcription factor subunits (p50, p65, c-Fos, Jun B, c-Jun, and CREB-1) were added. These findings indicate that cooperation between ERK- and cAMP-dependent pathways seems to be required to achieve the formation of an essential transcriptional factor complex for maximal H₂O₂-dependent chemokine modulation. Finally, experiments performed with actinomycin D suggest that H₂O₂-mediated MIP-1 mRNA up-regulation results from transcriptional control, whereas that of MIP-1, MIP-2, and monocyte chemoattractant protein-1 is due to both gene transcription activation and mRNA posttranscriptional stabilization.

	Introduction

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Hydrogen peroxide (H₂O₂), a reactive oxygen species (ROS)³, is an agent commonly produced during the oxidative stress generated in phagocytic cells as a defense mechanism against pathogen invasion (e.g., Leishmania donovani) (1). Moreover, H₂O₂, along with other ROS such as the superoxide anion (O₂^-), is generated during a variety of inflammatory conditions (2). In these cases, oxidative stress not only contributes directly to tissue damage but can also amplify inflammation by stimulating leukocyte recruitment through local chemokine induction (3, 4).

The direct contribution of H₂O₂ to the inflammatory response has been demonstrated in vivo by showing that its inhibition, and not that of O₂^-, decreases glucan-induced chemokine secretion and granuloma size formation in rats (5). These results suggest that H₂O₂ could be responsible for local chemokine expression and the development of inflammation. This in vivo evidence is further supported by numerous in vitro studies, which show the capacity of H₂O₂ to modulate chemokine expression in a variety of cell types. This cellular regulation includes the induction of IL-8 and monocyte chemoattractant protein (MCP)-1/CC chemokine ligand (CCL)2 in human epithelial (6), synovial (7), and endothelial cells (5), as well as that of macrophage (M)-inflammatory protein (MIP)-1/CCL3 (8) and MIP-2/CXC chemokine ligand 2 (3, 9) in rat alveolar M.

Recently, the concept of H₂O₂-induced tissue injury has been revised with the appreciation that this molecule can act as second messenger for signal transduction and thus affect proinflammatory gene expression. It has been demonstrated that H₂O₂ has the ability to inhibit protein tyrosine phosphatase activity (10). Furthermore, substantial evidence indicates that H₂O₂ leads to the activation of various serine-threonine and tyrosine protein kinases including protein kinase C (PKC) (11), as well as the mitogen-activated protein kinases (MAPK) extracellular signal-regulated kinases (ERK) 1 and 2, p38, and c-Jun amino-terminal kinase (12). However, only H₂O₂-induced PKC activation has been associated with chemokine expression (13).

In addition to its kinase-activating role, several studies show that H₂O₂ directly regulates the activity of various transcription factors, including CREB in astrocytes (14) and glial cells (15), as well as the reduction-oxidation (redox)-sensitive transcription factors NF-B (16, 17) and AP-1 (18, 19) in a variety of cell types. Of interest, activation of AP-1 and NF-B transcription factors has been associated with ROS-dependent chemokine induction. In vivo, the activation patterns of AP-1 and NF-B were found to correlate temporally with an increase in pulmonary MCP-1 levels in response to oxidative stress (20). In vitro, MCP-1 gene expression was shown to involve H₂O₂-induced AP-1 activity in endothelial cells, (21); IL-8 mRNA increase was linked to H₂O₂-dependent binding of AP-1 to the IL-8 promoter in epithelial cells (22) and transcriptional regulation of MIP-2 by H₂O₂ was associated with NF-B binding activity to the MIP-2 promoter in M (9). Although this redox regulation appears to occur at the transcriptional level and to be cell-type specific (18), the exact molecular mechanisms involved are largely unknown.

In the present study, we were interested in elucidating the transcriptional and posttranscriptional mechanisms whereby H₂O₂ mediates chemokine expression in murine M, a cell type that appears to be a potential source of chemokines in response to oxidative stress (3). We demonstrate that H₂O₂ increases mRNA levels of several chemokines (MIP-1, MIP-1, MIP-2, and MCP-1), activates the ERK pathway, and enhances DNA binding activity of various transcription factors (NF-B, AP-1, and CREB). Taken together, our results allow us to propose a multistep, multifactor model for H₂O₂-dependent chemokine regulation in M, which involves protein kinase activity, NF complex assembly, as well as chemokine gene transcription activation and mRNA transcript stability.

	Materials and Methods

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Reagents

H₂O₂ (3% v/v) and actinomycin D were purchased from Sigma-Aldrich (St. Louis, MO). Isotopes [-³²P]dUTP (3000 Ci/mmol) and [-³²P]dATP (3000 Ci/mmol) were obtained from ICN Pharmaceuticals (Montreal, Quebec, Canada). Specific inhibitors BAY 11-7082 and MDL-12,330A hydrochloride were purchased from Biomol Research Laboratories (Plymouth Meeting, PA). Apigenin and PD 98059 were obtained from Calbiochem (San Diego, CA).

Cell and culture conditions

The murine M cell line B10R, derived from the bone marrow of B10A.Bcgr (B10R) mice (23), was kindly provided by Dr. D. Radzioch (McGill University, Montreal, Canada). Cells were maintained in DMEM (Life Technologies, Rockville, MD) supplemented with 10% heat-inactivated FBS (HyClone Laboratories, Logan, UT) plus 100 µg/ml streptomycin and 2 mM L-glutamine at 37°C and 5% CO₂.

RNase protection assays (RPA)

mRNA expression studies were performed using an RPA kit (Riboquant; BD PharMingen, San Diego, CA), as we previously described (24). Total RNA was isolated from stimulated cells with TRIzol reagent (Life Technologies) according to the manufacturer’s protocol. Multiprobe mCK-5, which contains templates for the murine chemokines RANTES, MIP-1, MIP-1, MIP-2, IFN--inducible protein 10, MCP-1, and T cell activation gene 3, and the housekeeping genes ml-32 and GAPDH, was labeled with [-³²P]dUTP using T7 RNA polymerase. Labeled probe (3 x 10⁵ cpm) was allowed to hybridize with 10 µg of total RNA for 16 h at 56°C. mRNA probe hybrids were treated with RNase A, and extracted with phenol-chloroform. Protected hybrids were resolved on a 5% denaturing polyacrylamide sequencing gel and exposed to radiographic film overnight at -80°C. Laser densitometry was performed using an Alpha Imager 2000 digital imaging and analysis system (Alpha Innotech, San Leandro, CA).

Preparation of nuclear extracts

Cell stimulation was terminated by the addition of ice-cold PBS, and nuclear extracts were prepared according to the microscale preparation protocol (25). In brief, sedimented cells were resuspended in 400 µl of cold buffer A (10 mM HEPES (pH 7.9), 10 mM KCl, 1.0 mM DTT, and 0.5 mM PMSF). After 15 min on ice, 25 µl of 10% igepal (v/v) (Sigma-Aldrich) were added, and the lysate was vortexed for 10 s and centrifuged for 30 s at 12,000 x g. The supernatant was discarded and the cell pellet was resuspended in 100 µl of cold buffer B (20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and 1 mM PMSF). Cells were then rocked vigorously at 4°C for 15 min. Cellular debris was removed by centrifugation at 12,000 x g for 5 min at 4°C, and the supernatant was stored at -80°C until used.

EMSA

EMSA was performed using 6 µg of nuclear extract. Protein concentrations were determined using the commercial BCA Protein Assay Reagent (Pierce, Rockford, IL). Nuclear extracts were incubated for 20 min at room temperature in 1.0 µl of binding buffer (100 mM HEPES (pH 7.9), 40% glycerol, 10% Ficoll, 250 mM KCl, 10 mM DTT, 5 mM EDTA, and 250 mM NaCl), 2 µg of poly(dI-dC) and 10 µg of nuclease-free BSA (fraction V; Sigma-Aldrich) containing 1.0 ng of radiolabeled dsDNA oligonucleotide. dsDNA (100 ng) was end-labeled using [-³²P]dATP and T4 polynucleotide kinase (New England Biolabs, Beverly, MA). This mixture was incubated for 20 min at room temperature, and the reaction was stopped using 5 µl of 0.2 M EDTA. The labeled oligonucleotide was extracted with phenol/chloroform and passed through a G-50 spin column. The dsDNA oligonucleotides (Santa Cruz Biotechnology, Santa Cruz, CA), used as either probes or competitors, were as follows: consensus binding site for AP-1 c-Jun homodimer and Jun/Fos heterodimeric complexes, 5'-CGCTTGATGACTCAGCCGGAA-3'; consensus binding site for CREB of the CREB/activating transcription factor family, 5'-GAGATTGCCTGACGTCAGAGAGCTAG-3'; consensus binding site for NF-B/c-Rel homodimeric and heterodimeric complexes, 5'-AGTTGAGGGGACTTTCCCAGGC-3'; and consensus binding site for Sp1, 5'-ATTCGATCGGGGCGGGGCGAGC-3'. The oligonucleotides containing NF-B or CREB binding sites of the murine chemokine promoters were synthesized in our laboratory as follows: NF-B/MIP-1 5'-GTGCTTAAAATTTTCCCTCCTCAC-3' (26), NF-B/MIP-2 5'-GAGCTCAGGGAATTTCCCTGGTCC-3' (27), NF-B/MCP-1 5'-AAGGGTCTGGGAACTTCCAATACTGC-3' (28), and CREB/MIP- 5'-CTCGATGCCATGACATCATCTTTAC-3' (29). DNA-protein complexes were resolved from free-labeled DNA by electrophoresis in native 4% (w/v) polyacrylamide gels containing 50 mM Tris-HCl (pH 8.5), 200 mM glycine, and 1 mM EDTA. The gels were subsequently dried and autoradiographed. Cold competitor assays were conducted by adding a 100-fold molar excess of homologous unlabeled oligonucleotides of the various labeled dsDNA probes. Supershift assays were performed by preincubation of nuclear extracts with 2 µg of polyclonal Abs against p65 (Rel A), p50, c-Fos, Jun B, c-Jun, CREB-1, or Sp1 obtained from Santa Cruz Biotechnology, in the presence of all components of the binding reaction described above for 1 h at 4°C.

Western blotting

Cells were collected following stimulation and lysed in cold buffer containing 20 mM Tris-HCl (pH 8.0), 0.14 M NaCl, 10% glycerol (v/v), 1% igepal (v/v), 25 µM nitrophenyl guanidinobenzoate, 10 µM NaF, 1 mM sodium orthovanadate (Vi), and 25 µg/ml leupeptin and aprotinin. The lysates (20 µg/lane) were subjected to SDS-PAGE, and the separated proteins were transferred onto a polyvinylidene difluoride membrane (Millipore, Bedford, MA) as we previously described (24). After a 1-h blocking period in TBST containing 5% milk, the membranes were incubated overnight in TBST/5% BSA at 4°C with one of the following rabbit polyclonal Abs purchased from New England Biolabs: phospho-ERK1/ERK2 (Thr²⁰²/Tyr²⁰⁴), ERK1/ERK2, phospho-MAPK kinase (MEK)1/2 (Ser^217/221), MEK1/2, phospho-CREB (Ser¹³³), and CREB. Proteins were then detected with an anti-rabbit HRP-conjugated goat Ab (Affini-pure; Jackson ImmunoResearch Laboratories, West Grove, PA) and subsequent visualization by ECL (ECL Western blotting detection system; Amersham, Arlington Heights, IL).

	Results

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H₂O₂-induced chemokine mRNA up-regulation in murine M

Given that chemokine expression is regulated primarily at the gene transcription level (18) and because it has been suggested that M constitute a potential source of chemokines in response to oxidative stress (3), we investigated the direct effect of H₂O₂ on chemokine mRNA induction in the murine M cell line B10R. At first, cells were stimulated for 2 h with increasing doses of H₂O₂ (100–750 µM), and chemokine mRNA levels were monitored by RPA. As shown in Fig. 1A, we detected a significant and dose-dependent increase of chemokine mRNA expression over negative control for MIP-1 (10-fold), MIP-1 (3-fold), MIP-2 (20-fold), and MCP-1 (4-fold) when maximal H₂O₂ concentrations were added. In addition, kinetic analyses were performed to determine the incubation time needed to obtain maximal chemokine modulation with an intermediate dose of H₂O₂ (500 µM). As depicted in Fig. 1B, increased chemokine mRNA levels were detected very rapidly (1 h poststimulation), were maximal after 2 h, and transiently decreased over an 8-h period. Subsequent experiments were thus conducted, stimulating cells for 2 h with 500 µM H₂O₂.

View larger version (46K): [in this window] [in a new window]   FIGURE 1. Dose-response and kinetic analyses of H2O2-inducible chemokine mRNA up-regulation in murine M. Cells were treated with either increasing doses of H2O2 (100–750 µM) for 2 h (A) or 500 µM of H2O2 over an 8-h period (B) and chemokine mRNA expression was monitored by using a mCK5 multiprobe RPA system (left panels). Densitometric quantification of chemokine mRNA levels over negative control after normalization to GAPDH (right panels). Results are representative of one of three independent experiments.

Role of the ERK pathway on H₂O₂-mediated M chemokine mRNA expression

Having demonstrated that H₂O₂ was a potent chemokine inducer in M, we were next interested in determining which second messengers were involved in this cellular regulation. Previous studies have suggested the implication of the ERK pathway in chemokine induction in response to proinflammatory stimuli (30); however, activation of ERK1/ERK2 in response to H₂O₂ has not been reported yet in M. As illustrated in Fig. 2, following cell stimulation with H₂O₂, we observed a rapid and transient phosphorylation of MEK1/2 (Fig. 2A) and of ERK1/ERK2 (Fig. 2B). Thereafter, to investigate the involvement of the ERK pathway on the H₂O₂-mediated M chemokine mRNA induction, cells were treated with increasing doses of specific inhibitors directed against either MEK1/2 (PD 98059) or ERK1/ERK2 (Apigenin) before H₂O₂ stimulation. As depicted in Fig. 3A, low doses of PD 98059 (5 µM) caused a considerable decrease of chemokine induction (35% for MIP-1, 45% for MIP-1, 30% for MIP-2, and 25% for MCP-1), whereas doses of 10–20 µM totally inhibited the mRNA expression of all four chemokines. In correlation with these observations, cells incubated with intermediate doses of Apigenin (10 µM) showed a marked reduction of their chemokine transcripts (Fig. 3B) (55% for MIP-1, 45% for MIP-1, and 80% for MIP-2 and MCP-1), whereas maximal inhibitor concentrations (40 µM) completely abrogated mRNA expression. Altogether, this set of experiments suggests that the ERK pathway seems to play an important role in the H₂O₂-mediated M chemokine modulation.

View larger version (51K): [in this window] [in a new window]   FIGURE 2. Time course of H2O2-mediated MEK1/2 and ERK1/ERK2 phosphorylation. Protein lysates from H2O2-stimulated M over an 8-h period were subjected to Western blotting, and MEK1/2 (A) and ERK1/ERK2 (B) phosphorylation status was revealed using phospho-MEK1/2 and phospho-ERK1/ERK2 Abs. Equal protein levels were verified by using MEK1/2 and ERK1/ERK2 Abs. Results are representative of one of three separate experiments.

View larger version (50K): [in this window] [in a new window]   FIGURE 3. Effect of specific inhibitors of the ERK pathway on H2O2-dependent chemokine mRNA induction. Cells were treated (1 h) with either PD 98059 (A) or Apigenin (B) before H2O2-stimulation (2 h) and their effect on chemokine induction was evaluated by RPA (left panels). Integrated density values of chemokine mRNA levels normalized to GAPDH (right panels). Results are representative of one of three independent experiments. Nil: untreated (), H2O2 ± PD 98059 or Apigenin ().

Identification of H₂O₂-activated transcription factors involved in M chemokine regulation

It is well documented that H₂O₂ appears to be a physiological activator of the transcription factor NF-B in a variety of cells including murine M (16). However, in M, this activation has been linked only to H₂O₂-mediated rat MIP-2 transcription (9). Based on these observations, we initially evaluated whether this NF was activated in H₂O₂-stimulated B10R M. As shown in Fig. 4A, NF-B nuclear translocation was already increased 30 min following H₂O₂ treatment and was maximal 2 h poststimulation. To define the nature of the H₂O₂-induced NF-B complex, supershift assays were performed using Abs directed toward p50 and p65, two ubiquitous members of the NF-B family. As illustrated in Fig. 4B, the complex binding was diminished and partially supershifted by the anti-p50 Ab and almost completely abrogated by the anti-p65 Ab. These data indicate that H₂O₂ activates the DNA binding of both p50 and p65 NF-B subunits in murine M. To address the question whether H₂O₂ was not only leading to NF-B nuclear translocation but also to its binding to one or more chemokine genes, nuclear extracts from H₂O₂-treated cells were incubated with specific oligonucleotides containing the NF-B binding sites present in the murine MIP-1, MIP-2, and MCP-1 promoters. Interestingly, we observed that H₂O₂ stimulation led to the binding of NF-B to all chemokine genes examined (Fig. 4C).

View larger version (59K): [in this window] [in a new window]   FIGURE 4. H2O2 induces NF-B nuclear translocation and binding to several chemokine promoters in murine M. A, Nuclear extracts from cells either left untreated or stimulated with H2O2 for different time periods (0–8 h) were incubated with a -32P-labeled NF-B probe and were subjected to EMSA. Binding specificity was tested by adding to nuclear extracts from 2-h-treated cells a 100-fold molar excess of cold NF-B oligonucleotide. B, For supershift assays, nuclear extracts from cells treated with H2O2 (2 h) were incubated or not with specific Abs against the p50 and p65 NF-B isoforms for 1 h before EMSA. C, EMSA analysis was performed as described in A, but this time nuclear extracts were incubated with one of three NF-B probes specific for the murine MIP-1, MIP-2, or MCP-1 promoters. Binding specificity was tested by adding to nuclear extracts from 2-h-treated cells a 100-fold molar excess of either a cold NF-B/MIP-1, MIP-2, or MCP-1 oligonucleotide or a nonspecific Sp1 probe. These results are representative of one of three separate experiments.

View larger version (43K): [in this window] [in a new window]   FIGURE 5. Role of NF-B on M chemokine mRNA expression in response to H2O2. Nuclear extracts from either untreated or H2O2-stimulated cells (2 h) pretreated or not with Apigenin or PD 98059 (1 h) were incubated with a labeled NF-B oligonucleotide and subjected to EMSA (A). Either a consensus NF-B probe (B) or a probe containing a NF-B binding site in the murine MIP-2 promoter (D) were incubated with nuclear proteins from cells treated with BAY 11-7082 (1 h) before H2O2 stimulation (2 h), and EMSA were performed. Binding specificity was tested by adding to nuclear extracts from 2-h-treated cells a 100-fold molar excess of cold NF-B oligonucleotide. Total RNA was extracted from M stimulated, as described in the previous experiment, and chemokine mRNA modulation was monitored by RPA (C). Nil: untreated (), H2O2 ± BAY 11-7082 (). These results are representative of one of three independent experiments.

View larger version (50K): [in this window] [in a new window]   FIGURE 6. AP-1 nuclear translocation is induced by H2O2 via the ERK pathway in murine M. Labeled AP-1 probe was incubated with nuclear extracts from cells either nonstimulated or treated with H2O2 for different time periods (0–8 h) and EMSA analysis was performed (A). Nuclear proteins from H2O2-stimulated cells pretreated or not with Apigenin (B) or PD 98059 (C) were subjected to EMSA following incubation with an AP-1 consensus probe. Binding specificity was tested by adding to nuclear extracts from 4-h-treated cells a 100-fold molar excess of cold AP-1 oligonucleotide. These results are representative of one of three separate experiments.

MIP-1

View larger version (48K): [in this window] [in a new window]   FIGURE 7. H2O2 induces CREB phosphorylation and binding to the murine MIP-1 promoter in M. A, Time course of CREB phosphorylation in response to H2O2 (0–60 min) was monitored by Western blot as described in Fig. 2. B, Following incubation with a labeled CREB consensus probe, nuclear proteins from H2O2-stimulated cells (0–8 h) were subjected to EMSA. Binding specificity was tested by adding to nuclear extracts from 2-h-treated cells a 100-fold molar excess of cold CREB oligonucleotide. C, EMSA analysis was performed as described in B, but this time nuclear extracts were incubated with a probe containing the CREB binding site present in the murine MIP-1 gene. Binding specificity was tested by adding to nuclear extracts from 2-h-treated cells a 100-fold molar excess of either a cold CREB/MIP-1 oligonucleotide or a nonspecific Sp1 probe. These results are representative of one of three independent experiments.

View larger version (36K): [in this window] [in a new window]   FIGURE 8. Effect of MEK1/2, ERK1/ERK2, and cAMP inhibitors on H2O2-dependent CREB activation and chemokine modulation. Nuclear extracts from either untreated or H2O2-stimulated cells (2 h) pretreated or not with Apigenin or PD 98059 (1 h) were incubated with a labeled CREB oligonucleotide and subjected to EMSA (A). Following MDL-12,330A treatment (1 h) and H2O2 stimulation (2 h), either nuclear proteins or total RNA was extracted. Next, CREB binding activity (B) and chemokine mRNA modulation (C) were monitored by EMSA and RPA, respectively. Nil: untreated (), H2O2 ± MDL-12,330A (). Results are representative of one of three separate experiments.

H₂O₂-induced transcription factor complex formation in murine M

Our previous results clearly indicated that H₂O₂ was able to induce nuclear translocation of three different transcription factors (NF-B, AP-1, and CREB), and suggested a potential role for all of them on the H₂O₂-dependent activation of M chemokine genes. In addition, the cooperative action among different transcription factors, such as NF-B and AP-1, has been suggested to be involved in the expression of several chemokines (34, 35). This previous evidence along with our data prompted us to address the question of whether NF-B, AP-1, and CREB were acting in concert through complex formation, which could in turn lead to the observed chemokine gene up-regulation. To this end, nuclear proteins extracted from H₂O₂-stimulated cells were incubated with the radiolabeled NF-B probe, either in the absence or in the presence of a 100-fold molar excess of one of three cold oligonucleotides containing binding sites for either NF-B, AP-1, or CREB (Fig. 9A). As expected, H₂O₂-induced NF-B complex binding was completely abrogated in the presence of a 100-fold molar excess of cold NF-B oligonucleotide. Of interest, NF-B complex binding was strongly reduced in the presence of a 100-fold molar excess of either cold AP-1 or CREB oligonucleotides. We next performed the same kind of experiment as described above, while incubating nuclear extracts in presence of either the radiolabeled AP-1 (Fig. 9B) or CREB (Fig. 9C) probes. As it was observed for NF-B, both the H₂O₂-enhanced AP-1 and CREB binding activities were abolished in presence of a 100-fold molar excess of their respective specific cold oligonucleotides, and were markedly diminished when one of the specific oligonucleotides for the other two transcription factors were added in a 100-fold molar excess. To further address the question whether H₂O₂ was able to induce transcription factor complex formation, supershift assays were performed by incubating nuclear extracts from H₂O₂-treated cells with specific Abs against some of the main members of the NF-B (p50 and p65), AP-1 (c-Fos, Jun B, and c-Jun), and CREB (CREB-1) families before reaction with either NF-B, AP-1, or CREB labeled probes. As shown in Fig. 10, we detected a significant decrease of NF-B and AP-1 binding activities in the presence of all Abs. In the case of CREB, its DNA binding activity was reduced following incubation with Abs against JunB, p50, or p65, and a partial supershift was observed in presence of CREB-1 Ab. In contrast, when either an Sp1 Ab or an Sp1 cold oligonucleotide (100x nonspecific competition) were added, no changes in the binding of NF-B, AP-1, or CREB were detected, confirming the specificity of our previous observations. Altogether, these results allow us to suggest that H₂O₂ leads to the formation of a transcriptional complex involving NF-B, AP-1, and CREB, which in turn seems to be necessary for maximal modulation of various M chemokine genes.

View larger version (47K): [in this window] [in a new window]   FIGURE 9. H2O2-induced AP-1, CREB, and NF-B complex formation in murine M. Nuclear proteins from H2O2-stimulated cells were incubated with the radiolabeled NF-B probe, either in the absence or in the presence of a 100-fold molar excess of one of three cold oligonucleotides containing binding sites for either NF-B, AP-1, or CREB (A). The same experiment was performed as described above, but this time nuclear extracts were incubated in presence of the radiolabeled AP-1 (B) or CREB (C) consensus probes. These results are representative of one of three independent experiments.

View larger version (83K): [in this window] [in a new window]   FIGURE 10. Abs against various NF-B, AP-1, and CREB family members lead to changes on H2O2-induced NF-B, AP-1, and CREB binding activities in M. Supershift assays were performed by incubating nuclear proteins from H2O2-treated cells with specific Abs against Jun B, c-Jun, c-Fos, p50, p65, CREB-1, or Sp1 for 1 h at 4°C before reaction with a NF-B, AP-1, or CREB labeled probe. Binding specificity was tested by adding to nuclear extracts from H2O2-treated cells a 100-fold molar excess of either a cold NF-B, AP-1, or CREB oligonucleotide or a nonspecific Sp1 probe. These results are representative of one of three independent experiments.

Transcriptional and posttranscriptional regulation of M chemokines by H₂O₂

Increased levels of mRNA may be the result of transcriptional and/or posttranscriptional mechanisms of regulation. To establish through which of them H₂O₂ leads to an increase of M chemokine mRNA expression, we performed commonly used transcriptional inhibitor actinomycin D assays as previously described (8). At first, B10R cells were stimulated with H₂O₂ for 2 h either in the absence or in the presence of actinomycin D (5 µg/ml), and chemokine mRNA levels were monitored by RPA. As shown in Fig. 11A, cotreatment with actinomycin D completely blocked the expression of all four chemokine transcripts, suggesting that H₂O₂-dependent chemokine up-regulation is at least in part transcriptionally regulated. Next, the contribution of changes in mRNA stability to chemokine induction by H₂O₂ was evaluated by measuring chemokine mRNA t_1/2. As depicted in Fig. 11B, in the presence of actinomycin D, chemokine mRNA from nonstimulated cells (control) decayed rapidly with a t_1/2 of 30 min for MIP-1 and MIP-2, and 2 h for MIP-1 and MCP-1. H₂O₂ treatment significantly increased the t_1/2 of MIP-1, MIP-2 and MCP-1 mRNA, with a t_1/2 >4 h, indicating posttranscriptional stabilization. In contrast, H₂O₂ did not lead to an enhancement of MIP-1 t_1/2. Altogether, these results suggest that the induction of MIP-1 mRNA by H₂O₂ results from transcriptional regulation, whereas that of MIP-1, MIP-2, and MCP-1 is due to both transcriptional activation of their genes and posttranscriptional stabilization of their mRNA transcripts.

View larger version (38K): [in this window] [in a new window]   FIGURE 11. Transcriptional and posttranscriptional regulation of M chemokines by H2O2. A, B10R cells were stimulated with H2O2 for 2 h either in the absence or in the presence of actinomycin D (AD; 5 µg/ml). Next, total RNA was extracted and chemokine mRNA levels were monitored by RPA (left panel). Densitometric quantification of chemokine mRNA induction over negative control after normalization to GAPDH (right panel). B, Cells were left untreated (control) or stimulated with H2O2 for 2 h. Then, actinomycin D was added for different time periods (0–4 h). Following total RNA isolation, RPA analysis was performed (left panel). Densitometric quantification of the decay of chemokine mRNA normalized to GAPDH (right panel). H2O2, ; control, . Results are representative of one of three independent experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Although the current data provide evidence that H₂O₂ can function as a signaling molecule to activate gene expression, the transductional mechanisms underlying chemokine regulation by this ROS remain largely unexplored. Thus, in the present study, we have investigated the link between H₂O₂-induced M chemokine mRNA expression and the activation of several signaling pathways (ERK and cAMP) and transcription factors (NF-B, AP-1, and CREB), which have been identified previously as targets for oxidative stress in various experimental contexts.

ERK1/ERK2 MAPK have been shown to be activated by H₂O₂ in a variety of nonphagocytic cells (12, 14, 43, 44), and, in M, this has been suggested indirectly by exposing cells to hyperoxia (45) or to oxidant-generating agents (46). In concert with these findings, we provide direct evidence showing that H₂O₂ strongly induces phosphorylation of ERK1/ERK2 as well as of their immediate upstream activator, MEK1/2 in M. Even though the precise targets through which H₂O₂ activates the ERK pathway remain to be established, a possible mechanism could be intracellular tyrosine phosphatase inhibition, because these proteins show redox-sensitive cysteine residues in their active sites (47), and previous studies have demonstrated that exogenously added H₂O₂ could transiently and reversibly inactivate phosphatase activity (10). Alternatively, it is also plausible that H₂O₂-mediated ERK activation may involve a Ras-dependent mechanism. In fact, recent observations have demonstrated that Ras is an important sensor of oxidative stress (48), and MAPK are known to be activated downstream of Ras (49). Further studies are required to shed light on this matter.

Several reports have linked the ERK pathway to chemokine induction by several proinflammatory stimuli (30). In response to oxidative stress, this has only been suggested indirectly by showing that angiotensin II-mediated MCP-1 induction could be inhibited by blocking either H₂O₂ generation or MEK1/2 activation (50). Extending these previous observations, our data clearly show that blockage of the ERK pathway at two different levels of the signaling cascade (MEK1/2 and ERK1/ERK2) completely abolished the H₂O₂-mediated M chemokine mRNA expression. These results are consistent with an ERK-dependent signaling mechanism of chemokine regulation by oxidative stress.

The NF-B transcription factor plays important roles in immune and stress responses, and H₂O₂ has been shown to act as an effective inducer of its activity (17). Although we and others have demonstrated that H₂O₂ is able to induce M NF-B activation, the upstream processes leading to this cellular regulation have not yet been clearly elucidated. PKC and intracellular calcium were identified as targets of H₂O₂; however, further studies indicated that neither of them was involved in the H₂O₂-inducible M NF-B activation (51). Analysis of possible alternative second messengers revealed to us that blockage of the ERK pathway considerably reduced the H₂O₂-dependent M NF-B nuclear translocation. In contrast to our findings, Janssen-Heininger et al. (44) reported that these MAPK seem to act as negative regulators of NF-B in H₂O₂-treated epithelial cells. A possible explanation for this discrepancy may lie in the fact that cells are generally subjected to a redox regulation, pro-oxidant in the cytosol and reductant in the nucleus (52), and because the equilibrium between oxidants and antioxidants within these compartments may be cell-type specific (43), the same signaling pathway may act in opposite ways in different cells. In this context, we propose a novel mechanism for positive regulation of H₂O₂-inducible NF-B activation through the ERK pathway. Even though our results suggest an important role for ERK1/ERK2 in the H₂O₂-dependent M NF-B activation, it needs to be determined whether they act directly or via intermediate kinases. Moreover, because we did not detect total abrogation of NF-B activation by using specific inhibitors against these MAPKs, it is possible that other kinases, such as the IB kinase complex, which is also activated by H₂O₂ (53), could also contribute to this regulation.

In addition to NF-B, we observed that H₂O₂ was also able to increase the DNA binding activity of another redox-sensitive factor, AP-1. Our results further indicated that the H₂O₂-dependent AP-1 nuclear translocation, as well as that of NF-B, seems to be under the control of the ERK pathway. These data, together with previous reports showing that, in response to proinflammatory stimuli, both NF-B and AP-1 activation and chemokine induction can be attenuated by blocking the ERK cascade (30, 54), allow us to suggest that H₂O₂ may induce M chemokine expression, at least in part, by activating NF-B and AP-1 via ERK-dependent pathways. Whether NF-B and AP-1 contribute to this cellular regulation directly by binding to the chemokine promoters and/or by interacting with each other remains to be confirmed; however, different lines of evidence seem to support both possibilities. On the one hand, NF-B binding sites were reported for the murine MCP-1 (28), MIP-1 (26), and MIP-2 (27) promoters. Even though no NF-B binding sites have been identified in the murine MIP-1 promoter, studies performed with p50-deficient mice indicate that NF-B is required for MIP-1 production (55). In response to H₂O₂, AP-1 was found to bind to the human MCP-1 (21) and IL-8 (22) genes in endothelial and epithelial cells, respectively. Similarly, NF-B was shown to bind to the MIP-2 promoter in rat alveolar M following H₂O₂ treatment (9), and in accordance with this finding, our EMSA analysis revealed that NF-B was able to bind to the murine M MIP-1, MIP-2, and MCP-1 promoters in response to H₂O₂. In contrast, physical and functional interactions between AP-1 and NF-B have been reported (56) and maximal induction of several chemokine promoters has been shown to be dependent on the cooperative interaction between these two NF (35). In agreement with these published data, we detected a strong decrease on both H₂O₂-dependent NF-B and AP-1 DNA binding activities by reciprocal competition for these binding sites. In addition, supershift assays revealed significant reduction in H₂O₂-inducible NF-B and AP-1 binding activities in the presence of specific Abs against some of the main members of both transcription factor families (p50, p65, JunB, c-Jun, and c-Fos), further suggesting the formation of NF complexes in response to this ROS.

Analysis of possible alternative second messengers for H₂O₂-mediated M chemokine induction indicated the potential involvement of cAMP-dependent pathways. Even though the exact mechanisms by which cAMP may contribute to this regulation are not completely understood, based on our results and those presented by others, a potential role for cAMP-dependent CREB activation via the ERK pathway can be proposed. We observed that both the H₂O₂-mediated chemokine induction and CREB binding activity were down-regulated by specific inhibitors of both the ERK and cAMP pathways. In agreement with our observations, Lee et al. (57) recently showed that cAMP potentiates H₂O₂-induced ERK phosphorylation, and others (14) have reported the involvement of the ERK pathway in CREB phosphorylation by H₂O₂. Moreover, because increases of cAMP-dependent CREB phosphorylation can occur in the absence of ERK activation (58), it is also possible that the noticed CREB phosphorylation in H₂O₂-treated M may be due to the activation of other cAMP-dependent pathways different from ERK1/ERK2, such as the protein kinase A cascade. Alternatively, in addition to activating CREB, cAMP could also contribute to the observed H₂O₂-mediated increase of mRNA chemokine levels by up-regulating other transcription factors such as AP-1, via the cAMP response element-dependent transcription of the immediate early gene, c-fos (59), and/or NF-B and AP-1 via the ERK pathway.

Functional binding sites for CREB have been identified only in the murine MIP-1 (29) and RANTES (32) promoters. In line with these findings, we showed that CREB was able to bind to the murine MIP-1 promoter in H₂O₂-treated M, suggesting a direct contribution for this NF to MIP-1 gene activation by H₂O₂. However, it is possible that CREB also contributes to the H₂O₂-inducible MIP-1, MIP-2, and MCP-1 mRNA up-regulation by interacting with other transcription factors, such as NF-B and AP-1. In fact, our EMSA experiments revealed that the H₂O₂-dependent CREB DNA binding activity was dramatically reduced in the presence of an excess of cold AP-1 or NF-B oligonucleotides. Likewise, both NF-B/DNA and AP-1/DNA complexes were strongly diminished when a cold CREB probe was added in excess. Supporting these observations, a significant decrease of CREB binding activity was detected when specific Abs against NF-B (p50, p65) or AP-1 (JunB) families were added. Similarly, both NF-B/DNA and AP-1/DNA complexes were strongly reduced in the presence of a CREB-1 Ab, suggesting the formation of NF complexes. This is in agreement with reports showing functional interactions between NF-B and CREB family members (60) and with others who have described the formation of dimers between members of the AP-1 and CREB families (61). Based on our results and taking into account the physical interactions reported between the NF studied as well as their binding sites identified in the murine chemokine promoters, we propose a hypothetical model for M chemokine transcriptional activation in response to H₂O₂, which involves the formation of a multiprotein NF complex necessary for maximal chemokine up-regulation (Fig. 12).

View larger version (16K): [in this window] [in a new window]   FIGURE 12. Hypothetical model of H2O2-mediated chemokine transcriptional activation in murine M. Following cell exposure to H2O2, ERK- and cAMP-dependent signaling pathways are activated, leading to the nuclear translocation and DNA binding of NF-B, AP-1, and CREB. These transcription factors seem to form a multiprotein complex that might adapt its spatial conformation to bind to the available specific binding sites present in the murine MIP-1, MIP-1, MIP-2, and MCP-1 promoters and in such a way lead to maximal M chemokine transcription activation.

In conclusion, our findings allow us to propose a multistep and multifactor mechanism for H₂O₂-mediated chemokine regulation in murine M, which seems to involve transcriptional and posttranscriptional control and to require the cooperation between ERK- and cAMP-dependent pathways leading to the formation of an essential transcriptional complex (NF-B, AP-1, and CREB) for maximal and fine tuning of chemokine gene expression. Given the important link between oxidant production and human disease, our work may contribute to the delineation of the signaling pathways regulated by H₂O₂, which might be essential in the control of diverse cellular events associated with inflammatory conditions.

	Acknowledgments

 
We thank Marc Bergeron for his careful editing of the manuscript.

	Footnotes

 
¹ This work was supported by grants from the Canadian Institutes in Health Research to M.O. M.O. is member of a Canadian Institutes in Health Research Group in Host-Pathogen Interactions. M.O. is a recipient of a Canadian Institutes in Health Research Investigator Award and is a Burroughs Wellcome Fund Awardee in Molecular Parasitology. M.J. is a recipient of a Ministère de l’Éducation du Québec PhD studentship.

² Address correspondence and reprint requests to Dr. Martin Olivier, Centre de Recherche en Infectiologie, Centre Hospitalier Universitaire de Québec, Pavillon du Centre Hospitalier de l’Université Laval (RC-709), 2705 Boulevard Laurier, Ste-Foy, Québec, Canada G1V 4G2. E-mail address: martin.olivier{at}crchul.ulaval.ca

³ Abbreviations used in this paper: ROS, reactive oxygen species; MCP-1, monocyte chemoattractant protein-1; M, macrophage; MIP, M-inflammatory protein; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; MEK, MAPK kinase; CCL, CC chemokine ligand; PKC, protein kinase C; redox, reduction-oxidation; RPA, RNase protection assay.

Received for publication May 9, 2002. Accepted for publication October 18, 2002.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Murray, H. W., D. M. Cartelli. 1983. Killing of intracellular Leishmania donovani by human mononuclear phagocytes: evidence for oxygen-dependent and -independent leishmanicidal activity. J. Clin. Invest. 72:32.
2. Halliwell, B., J. M. Gutteridge. 1990. Role of free radicals and catalytic metal ions in human disease: an overview. Methods Enzymol. 186:1.[Medline]
3. Driscoll, K. E., L. Simpson, J. Carter, D. Hassenbein, G. D. Leikauf. 1993. Ozone inhalation stimulates expression of a neutrophil chemotactic protein, macrophage inflammatory protein-2. Toxicol. Appl. Pharmacol. 119:306.[Medline]
4. Driscoll, K. E. 2000. TNF- and MIP-2: role in particle-induced inflammation and regulation by oxidative stress. Toxicol. Lett. 112–113:177.
5. Kilgore, K. S., M. M. Imlay, J. P. Szaflarski, F. S. Silverstein, A. N. Malani, V. M. Evans, J. S. Warren. 1997. Neutrophils and reactive oxygen intermediates mediate glucan-induced pulmonary granuloma formation through the local induction of monocyte chemoattractant protein-1. Lab. Invest. 76:191.[Medline]
6. Lakshminarayanan, V., D. W. Beno, R. H. Costa, K. A. Roebuck. 1997. Differential regulation of interleukin-8 and intercellular adhesion molecule-1 by H₂O₂ and TNF- in endothelial and epithelial cells. J. Biol. Chem. 272:32910.[Abstract/Free Full Text]
7. Sato, M., T. Miyazaki, T. Nagaya, Y. Murata, N. Ida, K. Maeda, H. Seo. 1996. Antioxidants inhibit TNF- mediated stimulation of interleukin-8, monocyte chemoattractant protein-1, and collagenase expression in cultured human synovial cells. J. Rheumatol. 23:432.[Medline]
8. Shi, M. M., J. J. Godleski, J. D. Paulauskis. 1996. Regulation of macrophage inflammatory protein-1 mRNA by oxidative stress. J. Biol. Chem. 271:5878.[Abstract/Free Full Text]
9. Shi, M. M., I. Chong, J. J. Godleski, J. D. Paulauskis. 1999. Regulation of macrophage inflammatory protein-2 gene expression by oxidative stress in rat alveolar macrophages. Immunology 97:309.[Medline]
10. Hecht, D., Y. Zick. 1992. Selective inhibition of protein tyrosine phosphatase activities by H₂O₂ and vanadate in vitro. Biochim. Biophys. Acta 188:773.
11. Konishi, H., M. Tanaka, Y. Takemura, H. Matsuzaki, Y. Ono, U. Kikkawa, Y. Nishizuka. 1997. Activation of protein kinase C by tyrosine phosphorylation in response to H₂O₂. Proc. Natl. Acad. Sci. USA 94:11233.[Abstract/Free Full Text]
12. Ishikawa, Y., M. Kitamura. 2000. Anti-apoptotic effect of quercetin: intervention in the JNK- and ERK-mediated apoptotic pathways. Kidney Int. 58:1078.[Medline]
13. Dong, W., P. P. Simeonova, R. Gallucci, J. Matheson, R. Fannin, P. Montuschi, L. Flood, M. I. Luster. 1998. Cytokine expression in hepatocytes: role of oxidant stress. J. Interferon Cytokine Res. 18:629.[Medline]
14. Rosenberger, J., G. Petrovics, B. Buzas. 2001. Oxidative stress induces proorphanin FQ and proenkephalin gene expression in astrocytes through p38- and ERK-MAP kinases and NF-B. J. Neurochem. 79:35.