Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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Originally published online as doi:10.1189/jlb.0603280 on January 23, 2004

Published online before print January 23, 2004
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(Journal of Leukocyte Biology. 2004;75:689-697.)
© 2004 by Society for Leukocyte Biology

Activation of phosphatidylinositol 3-kinase and c-Jun-N-terminal kinase cascades enhances NF-{kappa}B-dependent gene transcription in BCG-stimulated macrophages through promotion of p65/p300 binding

Zoulfia Darieva, Elena B. Lasunskaia1, Mariana N. N. Campos, Thereza L. Kipnis and Wilmar D. da Silva

Laboratório de Biologia do Reconhecer, Universidade Estadual do Norte Fluminense, Campos, Rio de Janeiro, Brazil

1Correspondence: Laboratório de Biologia do Reconhecer, CBB, Universidade Estadual do Norte Fluminense, Av. Alberto Lamego, 2000, Campos/RJ, 28013-600 Brazil. E-mail: elena{at}uenf.br


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The proinflammatory response of infected macrophages is an important early host defense mechanism against mycobacterial infection. Mycobacteria have been demonstrated to induce proinflammatory gene transcription through the Toll-like receptors, (TLR)2 and TLR 4, which initiate signaling cascades leading to nuclear factor (NF)-{kappa}B activation. The main transduction pathway responsible for NF-{kappa}B activation has been established and involves the MyD88, interleukin-1 receptor-associated kinase, tumor necrosis factor receptor-associated factor-6, NF-{kappa}B-inducing kinase, and inhibitor of {kappa}B kinase complex. The role of other kinase cascades triggered by mycobacteria in the NF-{kappa}B activation is less clear. We herein examine the role of the mitogen-activated protein kinases (MAPKs) and phosphatidylinositol 3-kinase (PI-3K) cascades in the expression of the bacillus Calmette-Guerin (BCG) mycobacteria-induced NF-{kappa}B-dependent genes, macrophage-inflammatory protein-2 (MIP-2) and inducible nitric oxide (NO) synthase. Specific pharmacological inhibition of the PI-3K, c-jun-N-terminal kinase (JNK), and to a smaller extent, p38 MAPK but not extracellular-regulated kinase (ERK), suppressed NF-{kappa}B-dependent reporter gene transcription and MIP-2 and NO secretion in BCG-induced RAW264.7 macrophages. A similar effect was obtained following molecular inhibition of JNK via JNK-interacting protein-1 overexpression. In addition, a kinase-dead mutant of MEK kinase-1, the up-stream regulator of JNK, also proved to be a potent inhibitor of NF-{kappa}B-reporter activity. The effect of inhibitors was mediated by the down-regulation of NF-{kappa}B transcription activity and without effecting its nuclear translocation. These data suggest an indirect mechanism of the NF-{kappa}B regulation by these kinases, probably through p65 phosphorylation and improved binding to the p300 transcription coactivator. The data obtained demonstrate that PI-3K, JNK, and p38 MAPK activation by mycobacteria enhance NF-{kappa}B-driven gene expression contributing to the proinflammatory macrophage response.

Key Words: RAW264.7 cells • MIP-2 • iNOS • signal transduction


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mycobacteria cause intensive inflammation in infected organisms, mediated by rapid macrophage activation, proinflammatory gene transcription, and subsequent production of a variety of cytokines, chemokines, adhesion molecules, and short-lived, free radicals, including nitric oxide (NO). Transcription of most of proinflammatory genes, including macrophage inflammatory protein-2 (MIP-2)/CXC chemokine ligand (CXCL)2, and inducible NO synthase (iNOS), is nuclear factor-{kappa}B (NF-{kappa}B)-dependent. The MIP-2 promoter contains two {kappa}B sequence motifs and one NF-interleukin (IL)-6-binding site [1 ]. The iNOS murine promoter enhancer region, in addition to two NF-{kappa}B consensus sites, contains NF-IL-6 and interferon regulator factor-1 response elements [2 ], and a crucial role of the NF-{kappa}B in MIP-2 [3 ] and NO [4 ] production by murine macrophages is well documented.

The main signaling pathway leading to NF-{kappa}B activation by mycobacteria has been established. The mycobacterial lipoproteins, lipoarabinomannan and peptidoglycan, trigger signal transduction through mammalian Toll-like receptor (TLR)2, cytosolic adaptor protein MyD88, kinases of the IL-1 receptor-associated kinase (IRAK) family, tumor necrosis factor receptor-associated factor-6 (TRAF6), and NF-{kappa}B-inducing kinase (NIK). Activation of the inhibitor of {kappa}B (I{kappa}B) kinase (IKK) complex (IKK{alpha} and IKKß) by NIK causes phosphorylation of the I{kappa}Bs, targeting them for ubiquitylation and degradation by the proteasome that liberates NF-{kappa}B/Rel (p65/p50) dimers for nuclear translocation and transactivation of a variety of immune-response genes [5 , 6 ].

Signaling events induced by whole mycobacteria or its components, liberated through secretion or as a result of bacterial lysis, could be even more complex, involving at least two signaling TLRs, TLR2 and TLR4 [7 ], and additionally, complement, integrin, and lectin receptors [8 ]. A study of the summary effects of the whole bacteria on the regulatory pathways and establishment of their consequent effects on transcription factor activation and new gene expression are of particular importance to determine the possible targets for pharmacological regulation of inflammation.

Recent studies have demonstrated that proinflammatory gene transcription is tightly controlled by the transcription coactivator, cyclic adenosine monophosphate-response element-binding protein (CREB)-binding protein (CBP), and its structural homologue, p300, containing histone acetyltransferase activity that plays a role in chromatin remodeling [9 ]. Interaction with the CBP/p300 appears to be necessary to optimize the transcriptional activity of inducible transcription factors, including NF-{kappa}B [10 ], and allow them to associate with basal transcriptional machinery [11 ].

The importance of RelA/p65 and CBP/p300 physical association for proinflammatory gene transcription was confirmed in a variety of a cell types [12 , 13 ]. Recently, CBP/p300 was demonstrated to be essential for enhanceosome assembly and TNF-{alpha} gene transcription in Mycobacterium tuberculosis-stimulated murine macrophages [14 ]. NF-{kappa}B, however, was not considered to be critical for the TNF-{alpha} gene transcription in this study; thus, RelA/p65 and coactivator interaction in mycobacteria-stimulated macrophages has not been characterized.

Mitogen-activated protein kinases (MAPKs) and phosphatidylinositol 3-kinase (PI-3K) cascade pathways also contribute to the transmission of extracellular signals that can finally result in direct or indirect phosphorylation of various transcription factors and alteration in gene expression [15 ]. Mycobacteria have been shown to activate PI-3K [16 ] and MAPK cascades, including extracellular-regulated kinase (ERK)1/2, p38 kinase, and stress-activated protein kinases, such as the c-Jun-N-terminal kinase (JNK) [17 , 18 ]. Activation of MAPKs leads to direct activation of a number of transcription factors, including c-Jun (activated by JNK), activating transcription factor-2 (by p38 and JNK), and ets-like protein-1 (by ERK1/2), possibly contributing to cytokine production interfering with the NF-{kappa}B signaling pathway. The role of the PI-3K and MAPK pathways in mycobacteria-induced NF-{kappa}B activation, DNA binding, transcriptional coactivator recruitment, and resulting transcriptional activity of NF-{kappa}B has not been clearly established.

Selective inhibition of individual kinase pathways is an important approach to determine the contribution of a distinct pathway to cellular response to stimuli. In this study, we used commercially available pharmacological or molecular inhibitors to evaluate the involvement of MAPK and PI-3K pathways in NF-{kappa}B activation, NF-{kappa}B-dependent gene transcription, transcriptional coactivator p300/RelA recruitment, and MIP-2 and NO production by bacillus Calmette-Guerin (BCG) mycobacteria-stimulated RAW264.7 macrophages.

The data obtained allowed us to conclude that BCG-activated PI-3K, JNK, and to a lesser extent, p38 MAPK pathways contribute to NF-{kappa}B transcriptional activity, probably by promoting NF-{kappa}B-coactivator association, thus increasing production of MIP-2 and NO proinflammatory mediators.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
MAPK kinase (MEK)1 inhibitor PD98059, p38 MAPK inhibitor SB203580, PI-3K inhibitor LY294002, and NF-{kappa}B inhibitor BAY11-7085 were purchased from Calbiochem (San Diego, CA). SP600125, JNK inhibitor, and SN50 NF-{kappa}B inhibitory peptides were obtained from BioMol (Plymouth Meeting, PA). The firefly luciferase reporter assay system was from Promega (Madison, WI), and the Superfect reagent used for the transfection experiments was purchased from Qiagen (Valencia, CA). [{gamma}-32P]Adenosine 5'-triphosphate (ATP; >3000 Ci/mmol) and enhanced chemiluminescence (ECL) assay kits were from Amersham Life Sciences (Little Chalfont, UK). The cell-culture reagents, medium, fetal bovine serum (FBS), L-glutamine, and antibiotics were obtained from Gibco-BRL (Grand Island, NY).

Antibodies
Antibody against NF-{kappa}B RelA, rabbit anti-p65 antibody (#1226, #1207), was a kind gift from Dr. Nancy Rice (NCI-Frederick Cancer Research and Development Center, MD). Rabbit anti-PI-3K regulatory subunit p85, p300 (N-15), ERK1 (K-23), JNK1 (FL), p38 (C-20), NOS2 (M-19), and phospho-specific (p)-Akt1 (Ser 473)-R antibodies, goat polyclonal anti-Akt 1/2 (N-19), and mouse monoclonal phospho-specific p-ERK (E-4), p-JNK (G-7), and p-p38 (D-8) as well as Protein A/G Plus agarose were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antiphosphotyrosine rabbit polyclonal antibody was obtained from Transduction Laboratories (Lexington, KY).

Cell culture and mycobacteria
The RAW264.7 murine macrophage line (ATCC TIB-71; American Type Culture Collection, Mannasas, VA) was cultivated in Dulbecco’s modified Eagle’s medium-F12 supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, and 50 µg/ml gentamicin. Mycobacterium bovis (BCG vaccine, Onco-BCG, Moreau strain, Copenhagen SEED# July 1978) was purchased from the Instituto Butantan (São-Paulo, Brazil), where the vaccine was produced for bladder cancer treatment. The vaccine suspension was stored at 4°C and used in the experiments before the expiration date recommended by the manufacturer. To obtain BCG mycobacteria whole cell lysate (WCL), vaccine suspension was washed twice with sterile phosphate-buffered saline (PBS), pH 7.4, and sonicated in a lysing buffer of 10 mM Tris-HCl, 100 mM NaCl, 0.5% sodium dodecyl sulfate (SDS), and 25 mM EDTA, pH 7.8. BCG lysate preparations were passed through a Detoxi-Gel column using sterile pyrogen-free water, stored in pyrogen-free vials, and reconstituted with sterile, pyrogen-free PBS. Evaluation of bacterial endotoxin was performed with the amebocyte lysate assay (E-Toxate kit; Sigma Chemical Co., St. Louis, MO) and was less than 1 pg/ml final concentrations in all experiments.

For the experiments, RAW264.7 cells were grown in 96-well plates at a concentration of 3 x 105 cells/ml in complete medium at 37°C in 5% CO2 for 18 h. The culture medium was then changed for the same medium without serum and incubated for an additional 18 h. Resulting macrophage monolayers were infected with viable BCG at a multiplicity of infection (MOI) of 1:5 macrophage/bacteria or were treated with 1 µg/ml BCG WCL and incubated at 37°C for various time periods before the analysis. In some experiments, cells were pretreated with inhibitors of NF-{kappa}B: SN50 (100 µg/ml) or BAY11-7085 (10 µM) or with the kinase inhibitors PD98059 (25 µM), SB203580 (10 µM), LY294002 (25 µM), or SP600125 (1 µM) for 60 min before BCG stimulation. To serve as a control, the same volume of the diluent dimethyl sulfoxide (DMSO) contained in kinase inhibitors was added to the cell culture.

Culture supernatants were collected and stored at –20°C for further chemokine or nitrite quantification. For the intracellular protein analysis, cells were grown in 75 cm2 culture flasks, treated as indicated, washed in ice-cold, sterile PBS, harvested by scrapping, and used for further immunoprecipitation and Western blot or electrophoretic mobility shift assay (EMSA) analysis.

Phagocytosis assay
The macrophage monolayers grown on glass coverslips were pretreated or not with kinase inhibitors for 1 h and infected with viable BCG mycobacteria at a MOI of 1:5. After incubation for 3 h at 37°C, the cells were washed intensively with PBS to remove unbound bacteria and were stained by the Ziehl-Nielsen method. A Zeiss Axioplan microscope (x1200) was employed to analyze mycobacteria association with macrophages. At least 200 cells were examined in randomly chosen fields and scored for internalized or bound bacteria. The percentage of cells having at least one or more associated mycobacteria was determined. In control cultures that were infected without kinase inhibitors, 80% of macrophages were shown to be associated with mycobacteria. Bacterial phagocytosis by kinase inhibitor-treated macrophages was expressed as a percentage of the phagocytosis value obtained in control cells (defined as 100%).

Chemokine and nitrite quantification
The MIP-2 concentration in the culture supernatants was measured by sandwich enzyme-linked immunosorbent assay (ELISA) using The Quantikine M ELISA kit specific for mouse MIP-2 (R&D Systems, Minneapolis, MN). Accumulation of nitrites was determined as described previously [19 ]. Briefly, 50 µl triplicates of culture supernatants were mixed in 96-well plates with 50 µl Griess reagent, consisting of a 1:1 mixture of 1% sulfanilamide in 2.5% phosphoric acid and 0.1% naphtylenediamine dihydrochloride in deionized water. The absorbance at 570 nm was measured with a microplate reader. Nitrite concentrations were calculated using a standard curve of sodium nitrite, and results were presented as micromoles of nitrite.

EMSA
Nuclear extracts (10 µg), prepared according to Dignam et al. [20 ] at the indicated time points after macrophage stimulation, were subjected to EMSA in a reaction buffer containing 10 mM Tris (pH 7.9), 1 mM EDTA, 1 mM dithiothreitol, 1 µg poly (dI-dC), 100 mM NaCl, and 10% glycerol in a final volume of 20 µl. Oligonucleotide probes containing a single copy of the NF-{kappa}B motif, 5'-AGTTGAGGGGACTTTCCCAGGC-3' (Promega), were end labeled with [{gamma}-32P]ATP, and 10,000 cpm of the probe was used for DNA-binding analysis. Binding reactions were electrophoresed on native 4% polyacrylamide gels in 0.5 x Tris-boric acid-EDTA buffer to separate bound and unbound DNA probe. A competitive assay was performed with a 50-fold molar excess of unlabeled probe.

Immunoprecipitation
RAW264.7 cell pellets were resuspended in lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 0.5% Triton X-100, 1 mM Na orthovanadate, 50 mM NaF, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml leupeptin, aprotinin, and pepstatin) for 10 min on ice. After preclearing the lysate with normal rabbit immunoglobulin G (IgG; Sigma Chemical Co.) and Protein A/G agarose (Amersham Pharmacia Biotech, Little Chalfont, UK), the WCL (~500 µg protein) was incubated with anti-p65/RelA (2 µg) or anti-PI-3K p85 (3 µg) antibodies or normal rabbit IgG in a total volume of 500 µl for 18 h at 4°C. Protein A/G agarose was then added, and the mixture was rotated for an additional 1 h. The immunoprecipitates were collected by centrifugation, washed six times with 1 ml lysis buffer without protease inhibitors, eluted with SDS-polyacrylamide gel electrophoresis (PAGE) sample buffer, resolved on 7.5% SDS-PAGE, and analyzed by Western blotting.

Western blotting analysis for the presence of particular proteins was performed on whole cell proteins extracted by lysis buffer or on nuclear or immunoprecipitated proteins from RAW264.7 macrophage experiments as indicated above. Protein was mixed 1/1 with 2x sample buffer (20% glycerol, 4% SDS, 10% 2-mercaptoethanol, 0.1% bromphenol blue, and 0.125 M Tris-Cl, pH 6.8), boiled at 95°C for 5 min, and then submitted to SDS-PAGE. Cell proteins were transferred to nitrocellulose, and resulting membranes were blocked with 5% milk and then treated with the primary antibody of interest and horseradish peroxidase-conjugated anti-mouse or rabbit IgG secondary antibody. Immunoreactive bands were developed using a chemiluminescent substrate, ECL Plus. Blots that were probed with phospho-specific antibodies were stripped as recommended in the ECL protocol and reprobed with antibodies specific for the particular protein of interest.

Plasmids
The NF-{kappa}B-dependent luciferase reporter plasmid containing five copies of a consensus NF-{kappa}B site linked to a minimal luciferase promotor and pCV-ß-galactosidase (ß-gal) control vector was obtained from Stratagene (La Jolla, CA). Vectors expressing JNK-interacting protein-1 (JIP-1), JNK inhibitor, and dominant-negative (DN)-MEK kinase-1 (MEKK1), and DN-Raf-1 mutant (Raf301) in an expression vector were described elsewhere [21 22 23 ]. All plasmids were prepared using Qiagen endotoxin-free plasmid DNA purification columns. DNA was eluted from the columns using LPS-free buffers, and contaminating LPS levels were found to be less than 2 pg/ml.

Transfection and reporter assays
RAW264.7 cells were plated at a density of 106 cells per six-well plate in complete medium. After 24 h of growth to 30–40% confluence monolayer, each well was washed with the serum-free medium, and the cells were transiently transfected using Superfect reagent according to the manufacturer’s instructions. Transfection mixtures containing 0.5 µg pNF-{kappa}B-luciferase plasmid were combined with expression vectors containing 1 µg DN-MEKK1, DN-Raf-1, JIP plasmids, or empty vector and with 1 µg pCV-ß-gal control plasmid. Media were changed 18 h after transfection, and the cells were incubated for an additional 24 h in the fresh medium containing serum. The following day, individual wells were left untreated or were stimulated with 1 µg/ml BCG WCL for 6 or 24 h. The medium was then removed and stored at –20°C until further analysis for MIP-2 or nitrite. The cells were washed with sterile PBS, lysed in a luciferase lysis buffer, and assayed for luciferase activity according to the manufacturer’s instructions (Promega). Luciferase activity per mg total protein transfected with a particular DNA construct was normalized to that measured in cells transfected with an empty vector according to ß-gal expression. All transfection experiments were performed in triplicate and were repeated at least three times using different plasmid preparations; the mean values of two representative experiments are shown.

Immunofluorescent analysis
RAW264.7 cells were transfected and incubated for 18 h as indicated. Then the cells were scrapped and cultured on glass coverslips for an additional 24 h. Following 1 h stimulation with BCG WCL (1 µg/ml), the cells were fixed for 20 min with ice-cold methanol, washed, blocked, and incubated for 1 h with anti-p65 antibody (#1207). Goat anti-rabbit phycoerythrin-conjugated Igs were used as a secondary antibody (Sigma Chemical Co.). Coverslips were mounted over N-propylgallate and observed in a confocal laser-scanning microscope (LCM-310, Zeiss, Thornwood, NY, x800).

Data analysis
The number of experiments analyzed is indicated in each figure. Statistical analysis was performed using the unpaired Student’s t-test, and differences were determined to be statistically significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
BCG mycobacteria induce NF-{kappa}B activation and NF-{kappa}B-dependent proinflammatory mediator expression in RAW264.7 macrophages
RAW264.7 cells were infected with viable BCG mycobacteria at a MOI 5:1/bacteria:macrophage or were treated with BCG WCL, 1 µg/ml, and monitored for NF-{kappa}B activation and MIP-2 and NO production up to 48 h post-infection.

Viable or lysed, BCG mycobacteria caused rapid, within 1 h, NF-{kappa}B activation evaluated by p65/RelA translocation and accumulation in nuclei (Fig. 1a ) and NF-{kappa}B–DNA-binding activity (Fig. 1b) . The main difference was in duration of NF-{kappa}B activation. Lysed bacteria provoked intensive but brief activation that started to decrease 24 h after the stimulation, whereas viable bacteria induced sustained NF-{kappa}B activity up to 48 h of observation.



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  		Figure 1. BCG induces NF-{kappa}B activation and NF-{kappa}B-dependent proinflammatory mediator production by RAW264.7 macrophages. The cells were infected by BCG mycobacteria at MOI = 5:1 (A) or were treated with BCG WCL (1 µg/ml; B) for the time indicated. The cells were then collected and tested for NF-{kappa}B activation and iNOS expression. Culture supernatants were analyzed for the presence of MIP-2 and NO2–. (a) Kinetics of the NF-{kappa}B p65 protein translocation to the nucleus. Nuclear extracts were obtained, and proteins (20 µg) were subjected to Western blotting. Membranes were then treated with anti-p65/RelA antibodies. (b) EMSA analysis of NF-{kappa}B–DNA-inding activity. Nuclear extracts (10 µg) were submitted to the reaction using [{gamma}-32P]-labeled NF-{kappa}B oligonucleotide. (c) MIP-2 and NO2– concentrations in culture supernatants of BCG-treated cells were measured by sandwich ELISA or Griess reaction, correspondingly. The data reflect the mean ± SD of six independent experiments. Supernatants of untreated cultures were negative for nitrite and presented less than 0.5 ng ml–1 MIP-2. (d) Kinetics of iNOS expression. Cytoplasmatic fractions (100 µg) were subjected to Western blotting. Resulting membranes were treated with anti-iNOS antibodies.

 
Induction of NF-{kappa}B-dependent, proinflammatory molecule expression by BCG was evaluated by measuring MIP-2/CXCL2 chemokine and nitrite concentrations in culture supernatants of BCG-treated macrophage cultures. Additionally, we examined the kinetics of iNOS expession by Western blotting. Lysed mycobacteria caused a rapid MIP-2 response within 3 h, rising to maximum 24 h after macrophage treatment. Macrophage response to the viable bacteria was somewhat delayed, gradually increasing chemokine concentration reach peak to 48 h (Fig. 1c) . Induction of iNOS expression started 8 h after the BCG treatment; however, significant nitrite production was detected only 24 h post-treatment, increasing to 48 h (Fig. 1d) .

In these treatments, specific inhibition of NF-{kappa}B activation with BAY11-7085, a chemical compound that has been shown to decrease NF-{kappa}B expression by inhibiting I{kappa}B{alpha} phosphorylation, or with SN50-inhibitory peptide preventing NF-{kappa}B-activated complex binding to DNA, almost completely abrogated MIP-2 and nitrite secretion (Fig. 2 ), confirming that the expression of these molecules was highly dependent from {kappa}B transcription factor activity.



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  		Figure 2. Inhibition of BCG-induced NF-{kappa}B activation by specific pharmacological inhibitors suppressed production of MIP-2 and NO by RAW264.7 cells. The cells were pretreated with NF-{kappa}B inhibitors, SN50 or BAY11-7085, for 1 h and then stimulated with BCG WCL for the time indicated. (A) EMSA analysis of NF-{kappa}B–DNA-binding activity. Culture supernatants were analyzed for the presence of MIP-2 (B) and NO2− (C). The data demonstrate the mean ± SD of three independent experiments. C, Control.

 
BCG mycobacteria activate MAPKs and PI-3K/Akt pathways in RAW264.7 macrophages
We treated RAW264.7 cells with viable or lysed BCG and evaluated the kinetics of ERK, JNK, and p38 MAPK activation during the 4-h time period post-treatment by Western blotting using antibodies to the phosphorylated forms of corresponding kinases (Fig. 3 ). To determine whether BCG can activate PI-3K, we examined the phosphorylation status of the p85 regulatory subunit of PI-3K in tyrosine and activation of protein kinase B/Akt, a downstream component of PI-3K signaling. Mycobacteria caused rapid induction of ERK1/2, already 15 min post-treatment, which reduced to basal level within 4 h. PI-3K activation was rapid but transient: p85 phosphorylation obtained within 15 min was not more observed after 1 h. A significant level of Akt kinase phosphorylation was distinguished within 30 min, whereas p38 MAPK and JNK were significantly activated 30–60 min post-treatment. These kinases maintained a high level of activity during all of the period of observation. The activation time courses of all the kinases studied stimulated with viable or lysed mycobacteria were similar (the data are shown for BCG WCL).



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  		Figure 3. BCG induces MAPK and PI-3K/Akt pathway activation in RAW264.7 macrophages. The cells were incubated with BCG WCL for the time indicated and were lysed, and equal amounts of total cellular proteins (20 µg/lane for ERK; 100 µg/lane for p38, JNK, Akt) were submitted to Western blotting (WB). For PI-3K activity analysis, cellular proteins (500 µg) were first immunoprecipitated (IP) by anti-p85 PI-3K antibodies. Resulting nitrocellulose membranes were probed with antibodies specific to phosphorylated or nonphosphorylated ERK, p38, JNK, and Akt. Anti-p85 PI-3K and antiphosphotyrosine (anti-pTyr) antibodies were employed to treat IP proteins.

 
Inhibition of BCG-induced PI-3K or MAPK activation differentially down-regulates NF-{kappa}B transcriptional activity and secretion of NF-{kappa}B-dependent, proinflammatory molecules
To study the role of PI-3K and MAPK activation in NF-{kappa}B-dependent, proinflammatory signaling, we first investigated the effect of selective pharmacological inhibitors, LY294002 [24 ], SB203580 [25 ], SP600125 [26 ], and PD98059 [27 ], known to inhibit PI-3K, p38 MAPK, JNK, and MEK1, upstream kinase to ERK, respectively, on NF-{kappa}B activation and MIP-2 and NO secretion by BCG-treated macrophages.

In preliminary experiments, we established concentrations of the inhibitors sufficient for suppression of corresponding kinase phosphorylation induced by BCG without affecting cell viability, which was monitored by trypan blue exclusion assay (data not shown). We also tested the effect of distinct kinase inhibition on mycobacterial uptake, as some kinase pathways were demonstrated to be involved into the macrophage phagocytosis [28 ]. Pretreatment of RAW264.7 cells with LY294002 or PD98059 for 1 h before being infected with mycobacteria for 3 h reduced the phagocytosis index by 55% and 20%, respectively, whereas other inhibitors had no effect on bacterial uptake (Fig. 4A ). To avoid the differences in bacterial internalization that possibly could affect the level of the proinflammatory response of macrophages treated with distinct inhibitors, we used lysed bacteria in our inhibitory experiments.



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  		Figure 4. Effects of pharmacological kinase inhibitors on phagocytosis of BCG mycobacteria and NF-{kappa}B- dependent, proinflammatory mediator production by RAW264.7 macrophages stimulated with lysed bacteria. (A) The cells were pretreated with PD98059 (PD), SB203580 (SB), LY294002 (LY), SP600125 (SP), or DMSO for 60 min and were then infected with viable BCG mycobacteria at MOI = 5:1 bacteria/cell for 3 h at 37°C. The percentage of mycobacteria-associated macrophages was determined using microscopy. Values of mycobacterial association by macrophages obtained in the presence of kinase inhibitors were expressed as a percentage of the values obtained in the absence of inhibitors (defined as 100%). (B) Effects of kinase inhibitors on nuclear p65/RelA translocation and NF-{kappa}B binding to DNA. The cells were pretreated as indicated in A and then stimulated with BCG WCL for 1 h. Nuclear extracts of macrophages were submitted to the Western blotting or to the EMSA reaction as indicated in the legend to Figure 1 . (C) Effects of kinase inhibitors on transcriptional activity of NF-{kappa}B. Forty-eight hours before cell treatment with inhibitors and BCG, the cells were transiently transfected with NF-{kappa}B/Luc reporter vector as indicated in Materials and Methods. After stimulation with BCG WCL for 6 h, the cells were lysed and tested for luciferase activity. Luciferase activity is presented as fold-induction over the nonstimulated control. Data are mean of values from three experiments (±SD) with assays in triplicate. (D) Effects of kinase inhibitors on proinflammatory mediator production by stimulated macrophages. Transfected and treated with kinase inhibitors, cells were stimulated with BCG WCL for 24 h and tested for MIP-2 and NO production. Data are mean of values from four experiments (±SD) with assays in duplicate. Asterisks indicate significantly different values (*, P<0.05; **, P<0.01; Student’s t-test) from those obtained with the cells stimulated by BCG without inhibitor.

 
We found that macrophage pretreatment with all the inhibitors tested suppressed NO synthesis induced by BCG (Fig. 4D) . Chemokine secretion was also significantly inhibited in the presence of inhibitors, with the exception of PD98059. The most pronounced inhibitory effect was reached by suppression of the PI-3K/Akt pathway, providing 70% inhibition of MIP-2 and 56% of NO production. Inhibition of JNK reduced secretion by 30% and 50%, respectively. Less potent was the inhibitor of p38 MAPK; it caused 17% inhibition of MIP-2 and 23% of NO production. Inhibitor of the MEK1/ERK pathway had differential effect: by 25% suppressed NO production and had no effect on MIP-2.

Analysis of BCG-induced NF-{kappa}B activation demonstrated that neither pharmacological inhibitors prevented p65/RelA nuclear translocation and binding to DNA (Fig. 4B) . On the contrary, treatment by LY294002 increased the NF binding to labeled oligonucleotides. In contrast to DNA binding, expression of a reporter gene driven by a minimal promoter containing three NF-{kappa}B elements was significantly suppressed in BCG-infected macrophages pretreated with kinase inhibitors, with exception of that treated with PD98059 (Fig. 4C) . The more profound inhibition of transcriptional activity of NF-{kappa}B, by 62% and 50%, was observed after suppression of PI-3K and JNK, respectively, which coincided with strong inhibition of MIP-2 and NO secretion from these macrophages. Weak inhibition of NF-{kappa}B/Luc activity, by 30%, in cells treated by the p38 MAPK inhibitor corresponded to slight suppression of MIP-2 and NO production by BCG-stimulated cells (Fig. 4D) . The effect of MEK/ERK inhibition on NF-{kappa}B activation and proinflammatory mediator production was contradictory. PD98058 had no effect on NF-{kappa}B cytoplasmatic or nuclear activity and on MIP-2 synthesis in BCG-stimulated RAW264.7 cells but by 25% suppressed NO secretion.

To evaluate the role of up-stream regulators of JNK in MEKK1/MEK4/JNK cascade and ERK in Raf-1/MEK1/ERK cascade in macrophage response to BCG, kinase-dead mutants of MEKK1 or Raf-1 (Raf301) vectors or expression plasmid encoding JIP-1, JNK inhibitor, were transiently cotransfected along with the NF-{kappa}B/Luc reporter plasmid to RAW264.7 cells before stimulation. Transfected cells were stimulated by BCG WCL for 1 h and examined for NF-{kappa}B translocation to the nuclei. Neither kinase inhibition prevented nuclear accumulation of p65/RelA induced by BCG (Fig. 5A ). In contrast to nuclear translocation, NF-{kappa}B/reporter activity was significantly reduced in JIP-1 or DN-MEKK1-transfected cells (Fig. 5B) . These cells, in a similar manner, produced decreased levels of MIP-2 and NO (Fig. 5C) , suggesting that MEKK1 could contribute to JNK activation, thus regulating NF-{kappa}B-dependent, proinflammatory mediator synthesis in BCG-stimulated cells. DN-Raf-1 kinase overexpression failed to inhibit NF-{kappa}B-driven reporter gene transactivation as well as MIP-2 and NO secretion by these cells.



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  		Figure 5. Effects of molecular inhibition of the kinase pathways on NF-{kappa}B activation and proinflammatory mediator production. (A) RAW264.7 cells were transiently cotransfected with pNF-{kappa}B/Luc and empty vector (b) or additionally, with DN-MEKK1 (c), DN-Raf-1 (d), or JIP cDNA (e), as indicated in Materials and Methods, and then were stimulated with BC6 WCL (b–d) or left untreated [(a) control cells transfected with pNF-{kappa}B/Luc and empty vector]. After 1 h incubation, the cells were fixed, permeabilized, and immunostained with anti-p65/RelA antibodies to examine NF-{kappa}B nuclear translocation using immunofluorescent microscopy. (B) Transfected as indicated, cells were stimulated for 6 h, lysed, and measured for luciferase activity. Fold-induction to the level of unstimulated cells is demonstrated. (C) Cell-culture supernatants were collected and tested for MIP-2 and NO production 24 h after stimulation. Data are mean of values from two representative transfection experiments performed in triplicate (±SD). Asterisks indicate values significantly different (*, P<0.05; **, P<0.01; Student’s t-test) from those obtained from cells transfected with NF-{kappa}B/Luc without expression plasmid.

 
p65/RelA NF-{kappa}B subunit associates with p300 transcriptional coactivator in BCG-stimulated RAW264.7 cells that is promoted by activation of PI-3K and JNK pathways
As NF-{kappa}B-dependent transcription has been demonstrated to require the presence of p300/CBP coactivators in cytokine- or LPS-stimulated cells [12 , 13 ], we aimed to study the possibility of p65-p300 association in mycobacteria-stimulated macrophages and its regulation by MAPK and PI-3K cascades. Stimulated cells were lysed, and cellular proteins were immunoprecipitated by anti-p65/RelA antibody and analyzed by Western blotting. Consistent with previous results, p300 was present in immunoprecipitates in a BCG-dependent manner (Fig. 6 ), demonstrating a physical interaction of these proteins. Inhibition of PI-3K and JNK, but not ERK or p38 MAPK, prevented p65/p300 interaction, suggesting that the former kinase pathways could be involved in regulation of the transcriptional coactivator recruitment by the p65 subunit.



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  		Figure 6. Physical interaction of p65/RelA and p300 in BCG-treated RAW264.7 cells. The cells were pretreated or not by SP600125 (SP), PD98059 (PD), LY294002 (LY), or SB203580 (SB) kinase inhibitors for 1 h at 37°C and were then stimulated with BCG WCL for 1 h before lysis of the cells. Untreated cells were used as control (C). Whole cell extracts (500 µg protein) were immunoprecipitated (IP) with anti-p65 antibodies, submitted to 7.5% SDS-PAGE, and immunoblotted with anti-p300 and then with anti-p65 antibodies. Similar results were obtained in the two separate experiments. SP and LY pretreatment inhibits nuclear association of p300/CBP with p65. WB, Western blotting.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we aimed to determine whether MAPK and PI-3K pathways are involved in the regulation of NF-{kappa}B activation, NF-{kappa}B-driven gene expression, and therefore, into the MIP-2 and NO production.

BCG mycobacteria caused rapid nuclear translocation and DNA binding of activated NF-{kappa}B, presented, as it was earlier demonstrated, by a p65/p50 ubiquitous dimer [29 ]. The activation level induced by viable or lysed bacteria was similar within 24 h, although later, NF-{kappa}B activity was maintained only in macrophages infected with viable BCG. Synthesis of MIP-2 and NO proinflammatory molecules was in good accordance with NF-{kappa}B activation. The essential role of the NF in MIP-2 and NO production by BCG-stimulated RAW264.7 cells was confirmed by abrogation of the synthesis in the presence of SN50 and BAY11-7085, the specific inhibitors of NF-{kappa}B.

The kinetics of PI-3K/Akt and MAPK pathway activation by viable or lysed BCG mycobacteria was similar, at least within the period of observation.

Thus, there were no significant differences in the macrophage response to viable or lysed mycobacteria at the initial stages of activation within 24 h. Similarly, the comparable levels of nitrite secretion were observed in macrophages stimulated with viable or lysed BCG mycobacteria [30 ]. These results are in good accordance with recent data, demonstrating that mycobacterial viability was not an important factor for MAPK activation and subsequent TNF-{alpha} production by macrophages stimulated with viable or heat-killed M. tuberculosis or M. bovis BCG [31 ]. It seems that mycobacterial virulence factors [31 , 32 ], rather than bacterial viability and even integrity, determine the level of the initial macrophage activation by mycobacteria, although later, the presence of viable intracellular bacteria is important for the sustained macrophage activation.

To better learn the signaling events induced by mycobacteria in macrophages, we used pharmacological or molecular inhibitors to dissect the particular pathway.

First, we determined the effect of pharmacological kinase inhibition on mycobacterial phagocytosis, as the role of PI-3K in phagocytosis of opsonized microorganisms is well known [28 ]. Although our experiments were performed in serum-deprived medium to reduce the effect of serum on macrophage signaling, macrophage phagocytosis of nonopsonized mycobacteria was significantly suppressed by inhibitor of PI-3K, LY294002, and to a lesser extent, by PD98059, inhibitor of MEK/ERK, confirming the role of these kinases in phagocytosis. Nevertheless, the differences in mycobacteria uptake and MOI could additionally affect the level of macrophage activation, making difficult a comparison of the role of distinct kinases in NF-{kappa}B activation and subsequent gene expression. These data demonstrate a necessity of caution in interpretation of results obtained in the experiments using kinase inhibitors for the treatment of phagocytic cells. To prevent the effect of phagocytosis in a kinase-inhibitory test, we used lysed bacteria for macrophage stimulation in these experiments.

We found that pretreatment of RAW264.7 cells with kinase inhibitors suppressed MIP-2 and NO production induced by BCG. The most pronounced inhibitory effect was achieved via suppression of the PI-3K/Akt and JNK pathways. Less potent was the inhibitor of p38 MAPK, whereas inhibition of the MEK1/ERK pathway slightly reduced NO production and had no effect on MIP-2.

To verify whether the inhibitory effect observed was mediated by suppression of NF-{kappa}B, we studied its activity. Neither p65/RelA nuclear translocation nor DNA-binding activity was altered by any of the inhibitors tested. Conversely, PI-3K inhibition by LY294002 enhanced binding of NF-{kappa}B proteins. In contrast to DNA binding, NF-{kappa}B-dependent transactivation of the reporter gene was suppressed by all the inhibitors with exception of the ERK inhibitor, suggesting that examined pathways but not ERK pathway were important for p65/RelA phosphorylation known to be essential for NF-{kappa}B transcriptional activity [33 ].

To better characterize the pathways involved in regulation of MIP-2 and iNOS expression, we used molecular inhibition of kinases thought to be up-stream of the MAPKs. To suppress the MEKK1/MEK4/JNK signaling pathway, the cells were transfected with kinase-dead MEKK1 plasmid or expression plasmid encoding JIP-1 protein, a natural cytoplasmatic JNK inhibitor [21 ]. The DN-Raf-1 mutant, Raf301 [22 ], was used to inhibit down-stream MEK1/ERK. Inhibitory expression vectors were cotransfected along with NF-{kappa}B/Luc reporter plasmid to evaluate NF-{kappa}B transcriptional activity.

Nuclear translocation of BCG-activated NF-{kappa}B was not prevented by transfection, indicating that the studied kinases were not involved in an up-stream pathway leading to IKK-mediated phosphorylation of I{kappa}B{alpha}, its proteosome degradation, and liberation of the active p65/p50 dimer. MEKK1 was previously shown to be required for maximal activation of IKKs, as well as for NF-{kappa}B activity in cytokine-stimulated cells [34 ], but it is not likely that it is important for cytoplasmic NF-{kappa}B activation in mycobacteria-stimulated macrophages.

JIP-1- as well as DN-MEKK1-overexpressing cells presented significantly less luciferase gene expression in comparison with cells transfected with reporter plasmid only, confirming the data obtained with the pharmacological inhibitors and demonstrating the importance of the JNK pathway for NF-{kappa}B-dependent gene transcription. Inhibition of Raf-1-mediated signaling had no effect on NF-{kappa}B/Luc activation or MIP-2 and NO secretion. Nevertheless, the MEK1 inhibitor suppressed the latter, raising the possibility that ERK activation is not mediated by Ras/Raf-1 in BCG-stimulated RAW264.7 cells.

The data obtained demonstrate the involvement of PI-3K, JNK, and p38 MAPK pathways in the enhancement of transcriptional activity of NF-{kappa}B, suggesting a requirement of these kinase pathways for the expression of MIP-2 and iNOS genes at the level of NF-{kappa}B-driven transcription. These data, however, do not exclude the important contribution of the kinases studied for the activation of other transcription factors able to bind to enhancer elements in iNOS or MIP-2 gene promoters, providing a synergistic effect to gene transcription. Thus, ERK-mediated enhancement of NO production shown to be NF-{kappa}B-independent in our experiments suggests a role for this kinase in the regulation of synergistic transcription-factor activity or post-transcriptional events.

The mechanism of NF-{kappa}B activation by MAPK or PI-3K is not known, although the role of kinases in direct, or mediated by downstream kinase, phosphorylation of the p65 {kappa}B subunit is most likely. Appropriate phosphorylation of RelA/p65 enhances the transcription function of NF-{kappa}B [33 ], determining whether it associates in the nuclei with the transcriptional coactivator CBP/p300 promoting transcription [12 ]; otherwise, it binds histone deacetylase-1, suppressing NF-{kappa}B-dependent gene expression [35 ].

As we know, neither kinases studied were shown to phosphorylate p65 directly; however, it is still conceivable that some kinases, downstream to the PI-3K/Akt or p38 MAPK, are able to phosphorylate the NF-{kappa}B subunit. Previous studies have provided evidence that PI-3K/Akt signaling is involved in stimulation of the transcription function of NF-{kappa}B in some cell types [36 , 37 ]. The cytokine-activated PI-3K/Akt cascade has been shown to target nuclear p65 transactivation, inducing phosphorylation of the RelA/p65 in an Akt-dependent manner that could be mediated through IKK{alpha} phosphorylation [33 , 38 ] and p38 MAPK activation [39 ]. In the latter study, IKK was demonstrated to be responsible for p65 phosphorylation, whereas p38 indirectly stimulated the transactivation domain of p65 through a functional interaction with the transcriptional coactivator CBP/p300.

These data prompted us to verify the possible association of activated p65/RelA with p300 in BCG- stimulated cells and the involvement of PI-3K and MAPK in the regulation of this process. In protein complexes immunoprecipitated with the anti-p65 antibody from stimulated but not control cells, we detected the presence of p300 protein, demonstrating physical interaction of the coactivator with p65. Inhibition of PI-3K/Akt and JNK but not of MEK1/ERK or p38 MAPK suppressed the association of these two proteins. These data suggest that decreased p65-p300 association possibly mediated suppression of NF-{kappa}B transcriptional activity by LY294002 and SP600125 inhibitors. Impaired coactivator recruitment and consequent lack of histone or p65 acetylation were demonstrated to regulate gene transcription negatively [35 , 40 ].

In contrast to data obtained from cytokine-activated cells [39 ], p38 MAPK activation in BCG-stimulated macrophages had no effect on the p65-p300 interaction. Nevertheless, it is still conceivable that p38 kinase could contribute to NF-{kappa}B transactivation indirectly through phosphorylation of some target in basic transcription machinery [41 ] or histone H3 [42 ], facilitating transcription of NF-{kappa}B-dependent genes.

The regulatory role of the PI-3K/Akt pathway in p65 and p300 interaction could be mediated by p65 [33 , 39 ] or p300 [43 ] phosphorylation able to facilitate their binding. The role of JNK is most likely to be mediated by its main target, c-Jun, known to interact with p65 directly [44 , 45 ] or after binding to the proximal {kappa}B c-Jun-specific sites in gene promoters [46 ], thus providing a synergistic effect on gene transcription through mutual CBP/p300 binding [47 ]. In our system, the JNK inhibitor suppressed reporter gene transcription driven by NF-{kappa}B alone; therefore, the binding of other transcription factors to the luciferase promoter is unlikely, although the possibility of direct interaction of c-Jun with NF-{kappa}B cannot be discarded. A MIP-2 gene promoter, lacking c-Jun-binding sites, was recently demonstrated to be regulated synergistically by NF-{kappa}B, c-Jun, and CREB through direct binding of these transcription factors to the p65 subunit in murine macrophages stimulated with hydrogen peroxide [48 ]. A more detailed study is required to elucidate whether the same mechanism takes place in BCG-stimulated cells.

In summary, our findings provide evidence that the PI-3K/Akt, JNK, and p38 MAPK pathways are involved in the regulation of NF-{kappa}B-dependent gene transcription in murine macrophages stimulated by BCG mycobacteria. Most likely, kinase effects are mediated through promotion of the recruitment of the cointegrator molecules essential for a transcriptionally competent enhanceosome formation. Delineating the role of kinases, which might be inhibited by selective drugs, opens new transduction approaches for the treatment of the inflammation associated with mycobacterial infection.


   ACKNOWLEDGEMENTS
 
This work was supported by grants from Conselho Nacional de Pesquisa (CNPq-Rede TB) and Fundação de Apoio a Pesquisa do Estado do Rio de Janeiro (FAPERJ). Z. D. and E. B. L. contributed equally to this work. We thank Dr. Carlos Moreno for helpful discussions regarding the manuscript, Instituto Butantan for providing the vaccine, and Fernando C. Lopes for excellent technical assistance.

Received June 18, 2003; revised December 2, 2003; accepted December 12, 2003.


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