The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Díaz-Guerra, M. J. M.
Right arrow Articles by Boscá, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Díaz-Guerra, M. J. M.
Right arrow Articles by Boscá, L.
The Journal of Immunology, 1999, 162: 6776-6783.
Copyright © 1999 by The American Association of Immunologists

Negative Regulation by Protein Tyrosine Phosphatase of IFN-{gamma}-Dependent Expression of Inducible Nitric Oxide Synthase1

María J. M. Díaz-Guerra, Antonio Castrillo, Paloma Martín-Sanz and Lisardo Boscá2

Instituto de Bioquímica (Consejo Superior de Investigaciones Cientificas-UCM), Facultad de Farmacia, Universidad Complutense de Madrid, Madrid, Spain


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Treatment of cultured peritoneal macrophages with IFN-{gamma} resulted in tyrosine phosphorylation of I{kappa}B{alpha} and I{kappa}Bß, NF-{kappa}B activation, and expression of inducible NO synthase (iNOS). Since tyrosine phosphorylation of I{kappa}B{alpha} is sufficient to activate NF-{kappa}B in Jurkat cells, macrophages were treated with the protein tyrosine phosphatase inhibitor peroxovanadate (POV), which elicited an intense tyrosine phosphorylation of both I{kappa}B. However, this phosphorylation failed to activate NF-{kappa}B. Treatment with POV of macrophages stimulated with IFN-{gamma} or LPS potentiated the degradation of I{kappa}B{alpha} and I{kappa}Bß, the activation of NF-{kappa}B, and the expression of iNOS. Analysis of the iNOS gene promoter activity corresponding to the 5'-flanking region indicated that POV potentiates the cooperation between IFN-{gamma}-activated transcription factors and NF-{kappa}B. These results indicate that tyrosine phosphorylation of I{kappa}B is not sufficient to activate NF-{kappa}B in macrophages and propose a negative role for protein tyrosine phosphatase in the expression of iNOS in response to IFN-{gamma}.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Macrophage activation constitutes an important step in the onset of immune and inflammatory processes since these cells integrate multiple extracellular stimuli released by T lymphocytes and by molecules from pathogens, including viruses, bacteria, and fungi (1, 2). As a result of this activation, the macrophage releases additional cytokines involved in the immune response, as well as reactive nitrogen- and oxygen-derived molecules, which participate directly in cell killing (1, 2, 3, 4, 5). Synthesis of IFN-{gamma} constitutes a pivotal step in the process of macrophage activation. This cytokine is released mainly by T lymphocytes and triggers the transcription of several genes implicated in inflammation and cell adhesion (6, 7).

Signaling through the IFN-{gamma} receptor is initiated by ligand-induced tyrosine phosphorylation of Jak1 and Jak2, which in turn phosphorylate a unique tyrosine residue located in the cytoplasmic domain of the receptor, allowing Stat1{alpha} and -ß to interact with the receptor and to be phosphorylated by Jak activities. Phosphorylated Stat1 dimers translocate to the nucleus where they bind to cognate DNA sequences and influence the transcription of specific genes (6, 7, 8). Resetting of IFN-{gamma}-induced signaling is mediated through constitutive protein tyrosine phosphatase (PTP)3 activity associated with the receptor and acts as negative regulator of the process (8, 9, 10, 11).

The high output NO synthesis accomplished by inducible NO synthase (iNOS) constitutes an important event in the host defense and in the regulation of immune responses (2, 5, 12, 13, 14). In this context, IFN-{gamma}, acting in concert with bacterial products or with proinflammatory cytokines, potentiates the transcription of the iNOS gene in several types of cells (14, 15, 16). Indeed, in peritoneal macrophages, high doses of IFN-{gamma} may initiate the synthesis of NO through the engagement of response elements present in the promoter region of the iNOS gene (17, 18). The well-characterized 1.7-kb fragment of the 5'-flanking region of the murine iNOS gene contains at least 24 consensus sequences for the binding of transcription factors regulated by proinflammatory cytokines, including 10 copies of IFN-{gamma} response elements, 3 copies of the {gamma}-activated site (GAS), 2 copies of the IFN-{gamma}-stimulated response element (IRF1), and 2 copies of {kappa}B sites (17, 18, 19). The expression of iNOS in murine macrophages is strictly dependent on NF-{kappa}B activation, and therefore the engagement of this transcription factor could be predicted in the induction of iNOS when IFN-{gamma} is the unique stimulus (17, 18, 19).

Activated NF-{kappa}B complexes, in macrophages composed mainly of p50 and p65 subunits, are translocated to the nucleus in response to cell stimulation (20, 21, 22). This activation of NF-{kappa}B requires phosphorylation and degradation of the I{kappa}B proteins, and only recently have the serine/threonine kinases that phosphorylate I{kappa}B in specific serine residues that target the protein for ubiquitin conjugation and degradation by the 26S proteasome been identified (23, 24). Interestingly, tyrosine phosphorylation of I{kappa}B{alpha} is sufficient to activate NF-{kappa}B in human lymphoid cells, in the absence of proteolytic degradation, allowing the complex to translocate to the nucleus and bind to {kappa}B motifs in the DNA (25, 26). Keeping in mind the possibility of phosphorylation of I{kappa}B in tyrosine residues as a signal sufficient for NF-{kappa}B activation, we have investigated whether this mechanism is functional in peritoneal macrophages challenged with IFN-{gamma} and might contribute to the observed iNOS expression in these cells. Our data show a moderate expression of iNOS in response to IFN-{gamma}, accompanied by tyrosine phosphorylation of both I{kappa}B{alpha} and I{kappa}Bß. Inhibition of PTP with peroxovanadate (POV) induced an important tyrosine phosphorylation of both I{kappa}B proteins, but this was unable to activate NF-{kappa}B in murine macrophages. POV potentiated markedly the response to IFN-{gamma} in terms of iNOS expression, including a more rapid and greater NF-{kappa}B and Stat1 activation. These results suggest a negative role for PTP in the control of iNOS expression in the macrophage.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals

Cytokines were from Boehringer Mannheim (Mannheim, Germany). LPS from Escherichia coli and other reagents were from Sigma (St. Louis, MO). Serum and other cell culture reagents were from BioWhittaker (Walkersville, MD). Abs were from Santa Cruz Biotechnology (Santa Cruz, CA).

Cell cultures and treatments

Elicited peritoneal macrophages were prepared from 2-mo-old male mice 4 days after i.p. inoculation of 1 ml of sterile 10% thioglycollate broth (22). Cells were seeded at 1.5 x 106 in 6-cm plates and cultured with RPMI 1640 medium supplemented with 10% heat-inactivated FCS and antibiotics, at 37°C in an atmosphere of humidified 5% CO2. After incubation for 1 h, nonadherent cells were removed, the dishes were washed twice with PBS, and the remaining cells were cultured and stimulated for different periods of time in phenol red-free RPMI 1640 medium containing 1% FCS.

Jurkat T cells and RAW 264.7 macrophages (American Type Culture Collection, Manasses, VA; No. TIB-71) were grown in RPMI 1640 medium as indicated for peritoneal macrophages.

Preparation of POV

A fresh solution of POV was obtained by incubating 1 mM vanadate in PBS with 1 mM H2O2 for 10 min at 30°C (9, 10, 25). The mixture was treated with catalase (200 µg/ml) to remove the residual H2O2. This preparation of POV was used up to 30 min after preparation. Treatment of cells with catalase and H2O2 was used as a control of specificity of POV effects.

Plasmids

The 1753-bp HincII fragment corresponding to the 5'-flanking region of iNOS fused to a promoterless chloramphenicol acetyltransferase (CAT) reported gene (p1iNOS.CAT) (18, 22) was a generous gift from Drs. Q.-w. Xie and C. Nathan (Cornell University, New York, NY). Plasmids ({kappa}B)3ConA.CAT and ConA.CAT have three copies of the {kappa}B motif from the HIV long terminal repeat enhancer and the minimal promoter with no enhancer element of the conalbumin A promoter (used as a control), respectively, and have been previously described (27). Mutated {kappa}B sequences of the promoter (nucleotides -980 to +165) were generated by PCR using oligonucleotide primers in which two GG bases of the distal {kappa}B motif (position -971 to -961; p2iNOS(-,+).CAT vector), proximal {kappa}B motif (position -85 to -75; p2iNOS(+,-).CAT vector), or both (p2iNOS(-,-).CAT) were replaced by a CC pair, and were kindly given by Dr. T. J. Evans (28). These vectors were sequenced to ascertain their fidelity. Plasmids mutated in two conserved nucleotide positions of the GAS and IFN-stimulated response element (ISRE) motifs within the iNOS promoter were kindly provided by Dr. W. J. Murphy (15). A kSV2.CAT plasmid in which the CAT gene is driven by the SV40 early promoter and enhancer was used as a control in transfection assays (27).

Preparation of cytosolic and nuclear extracts

Protein extracts were prepared following the method of Schreiber et al., as described previously (29). Protein content was assayed using the Bio-Rad (Richmond, CA) detergent-compatible protein reagent. All steps of cell fractionation were carried out at 4°C.

Immunoprecipitation of proteins

Equal amounts of cytosolic protein extracts (100–200 µg) were incubated for 18 h at 4°C with Sepharose-immobilized rabbit anti-P-Tyr (PY20), anti-I{kappa}B{alpha} or anti I{kappa}Bß Abs with continuous rotation (27). After centrifugation, the agarose beads were washed five times with a large excess of buffer (10 mM HEPES (pH 7.9), 1 mM EDTA, 1 mM EGTA, 100 mM KCl, 1 mM DTT, 0.5 mM PMSF, 2 µg/ml aprotinin, 10 µg/ml leupeptin, 2 µg/ml N{alpha}-p-tosyl-L-lysine chloromethyl ketone, 5 mM NaF, 1 mM NaVO4, and 10 mM Na2MO4), with continuous rotation. The suspension was centrifuged and the beads were then mixed with 100 µl of 2x Laemmli sample buffer and heated at 80°C for 5 min. After centrifugation, the supernatant was size-fractionated in 10% SDS-PAGE.

Electrophoretic mobility shift assays (EMSAs)

The oligonucleotide sequences corresponding to the consensus NF-{kappa}B binding site (nucleotides -978 to -952) 5'-TGCTAGGGGGATTTTCCCTCTCTCTGT-3' (18, 22) of the murine iNOS promoter, or the motif corresponding to the Ly-6E GAS site (30) 5'-gtcATATTCCTGTAAGTG-3' were synthesized. Aliquots of 100 ng of these annealed oligonucleotides were end-labeled with Klenow enzyme fragment. A total of 5 x 104 dpm of the DNA probe were used for each binding assay of nuclear extracts as follows: 3 µg of protein were incubated for 15 min at 4°C with the DNA and 2 µg of poly(dl:dC), 5% glycerol, 1 mM EDTA, 100 mM KCl, 5 mM MgCl2, 1 mM DTT, and 10 mM Tris-HCl (pH 7.8) in a final volume of 15 µl. The DNA-protein complexes were separated on native 6% polyacrylamide gels in 0.5% Tris-borate-EDTA buffer (22, 27). Supershift assays were carried out after incubation of the nuclear extract with the Ab (0.5 µg) for 1 h at 4°C, followed by EMSA. Anti-p50 (human), anti-c-Rel (human), and anti-p65 (murine) Abs were from Santa Cruz Biotechnology.

Western blot analysis

Proteins (15 µg) were size-separated in minigels (7 cm) of 10% SDS-PAGE. I{kappa}B{alpha}, I{kappa}Bß, IRF1, and Stat1 were recognized by the corresponding Abs (Santa Cruz), and revealed following the ECL technique (Amersham). Autoradiographies were quantified by laser densitometry (Molecular Dynamics) and various exposition times were analyzed to ensure the linearity of the band intensities. At the end of the experiment, the membranes were treated with Ponceau S reagent to confirm the protein charge after blotting.

Transient transfection of RAW 264.7 macrophages and reporter assays

RAW 264.7 cells were transfected with 1,3-di-oleoyloxy-2-(6-carboxy-spermyl)-propylamide transfection reagent (Boehringer Mannheim) following the instructions of the supplier. After 6 h of culture the medium was aspirated and the dishes washed twice with incubation medium. The cells were maintained overnight with incubation medium supplemented with 2% FCS and followed by stimulation with different factors. After incubation for 24 h, the medium was aspirated and the cell layer washed twice with ice-cold PBS. Cells were scraped from the dishes, recovered by centrifugation, and submitted to three cycles of freezing and thawing. The cell extract was prepared by adding to each tube 0.2 ml of 0.25 M Tris-HCl (pH 7.8) at 4°C, followed by centrifugation at 12,000 x g for 10 min. The supernatant was heated at 65°C for 10 min and the CAT activity was measured by the synthesis of acetylated [14C]chloramphenicol following the TLC method (27). When luciferase activity was measured, cells were homogenized using the Promega (Madison, WI) luciferase assay kit assay and following the manufacturer’s instructions. Challenge of cells with an equal amount of plasmid in the absence of transfection reagent was unable to induce NO synthesis. The content of endotoxin in the plasmid preparations was below 35 pg/ml once in the cell culture, using the Limulus polyphemus test (Sigma).

Statistical analysis

Results are expressed as the mean ± SEM of the indicated number of experiments. Statistical significance was estimated using Student’s t test for unpaired observations. A probability value of less than 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
POV potentiates the expression of iNOS

Incubation of cultured macrophages with POV alone did not induce the expression of iNOS in these cells. However, POV potentiated markedly the synthesis of NO elicited by IFN-{gamma} (4.3-fold increase) and by LPS, although to a lesser extent (1.9-fold increase). When IFN-{gamma} and LPS acted in concert in promoting iNOS expression, the potentiation by POV was negligible, indicating that the contribution of PTP inhibition to iNOS expression is covered by the synergism between IFN-{gamma} and LPS signaling (Fig. 1GoA). The effect of POV on iNOS expression was specific since treatment of the cells with the doses of H2O2 and catalase used in its preparation was unable to modify the synthesis of NO elicited by IFN-{gamma} or LPS. When the iNOS protein levels were measured by Western blot, good agreement was observed between the band intensities and the amount of NOx- measured (Fig. 1GoB). The maximal potentiation of IFN-{gamma}-dependent NO synthesis by POV was obtained at 10–20 µM concentrations (Fig. 1GoC). Concentrations of POV higher than 40 µM were toxic to the macrophage, but not to other cells such as Jurkat T cells, which tolerated concentrations up to 200 µM without loss of viability (not shown). Fig. 1GoC also shows that POV by itself, assayed at concentrations up to 30 µM, was unable to increase NO synthesis. To determine the period of time at which POV potentiated the effect of IFN-{gamma}, macrophages were treated with 10 µM POV at different times with respect to IFN-{gamma} challenge (0 time), and NO synthesis was measured at 18 h. As Fig. 1GoD shows, treatment of cells with POV from 30 min prior to stimulation to 1 h after IFN-{gamma} addition resulted in the maximal potentiation, which decreased progressively up to 4 h, a time at which the effect of POV was completely abolished. These results suggest that POV enhances the effect of IFN-{gamma} in macrophages through the engagement of early pathways that participate in the process of iNOS expression.



View larger version (27K):
[in this window]
[in a new window]
  		FIGURE 1. POV synergizes with IFN-{gamma} or LPS in the expression of iNOS in peritoneal macrophages. Cultured peritoneal macrophages (1.5 x 106 cells) were prepared from thioglycollate-elicited mice and were treated with 10 µM of a recently prepared POV solution and 50 U/ml of IFN-{gamma}, 500 ng/ml of LPS, or both, and the amount of nitrate plus nitrite released to the medium was determined after 18 h of culture (A). At the end of the incubation period, cytosolic extracts were prepared and equal amounts of protein (15 µg) were analyzed by Western blot to determine the iNOS content using a specific Ab (B). The dose-dependent effect of POV on the potentiation of IFN-{gamma} (50 U/ml) was measured following the NO synthesis after 18 h of stimulation (C). The effect on NO synthesis (18 h) of POV addition from 30 min prior to stimulation to 8 h after IFN-{gamma} challenge (time 0) was determined (D). Results show the mean ± SEM of three experiments (A and C), or the mean of two experiments (D). *, p < 0.001 with respect to the incubation in the absence of POV.

 
POV does not activate NF-{kappa}B in macrophages but potentiates the effect of IFN-{gamma}

The expression of iNOS is very dependent on the extent of NF-{kappa}B activation (17, 18, 19), and previous reports described that POV induces an important activation of NF-{kappa}B in Jurkat T cells (25). As Fig. 2GoA shows, POV alone completely failed to activate NF-{kappa}B in macrophages using EMSA of the {kappa}B motif corresponding to the distal site in the murine iNOS promoter (22). However, under identical conditions, an important activation of NF-{kappa}B was observed in Jurkat cells (Fig. 2GoA, right). Treatment of macrophages for 1 h with POV and IFN-{gamma} increased the intensity of the bands corresponding to the NF-{kappa}B complexes, showing a potentiation of the action of IFN-{gamma}. The effect of POV on the NF-{kappa}B activity of cells treated with LPS was quantitatively less important. Analysis of the proteins involved in the formation of these NF-{kappa}B complexes revealed the presence of p50 dimers in the lower band and mainly p50.p65 heterodimers in the upper band. The distribution of p65 in cytosolic and nuclear extracts was determined in the same experiment, and an important translocation to the nucleus was observed in cells treated simultaneously with POV and IFN-{gamma} or LPS (Fig. 2GoB). Since NF-{kappa}B is transiently activated in activated macrophages (18, 22), the time course of the binding of proteins to the {kappa}B motif was investigated using EMSA, and following the intensity of the upper band. As Fig. 2GoC shows, POV by itself did not affect the binding but promoted an early potentiation in the response to IFN-{gamma}; however, the effect of POV was less effective in LPS-activated macrophages.



View larger version (52K):
[in this window]
[in a new window]
  		FIGURE 2. POV potentiates NF-{kappa}B activation in macrophages stimulated with IFN-{gamma}. Peritoneal macrophages were kept in culture and stimulated for 1 h with 10 µM POV, 50 U/ml of IFN-{gamma}, 500 ng/ml of LPS, or combinations of these. Nuclear extracts were prepared to determine NF-{kappa}B binding by EMSA. The specificity of the assay was confirmed by the displacement of the binding when a 20-fold excess of unlabeled oligonucleotide was added to the incubation of the nuclear extract with the probe (A, left). In addition to macrophages, nuclear extracts from Jurkat cells treated under identical conditions were prepared and analyzed. Supershift assays were performed by incubating the nuclear extracts with 0.5 µg of the indicated Ab followed by EMSA (A, right). The amount of p65 in the cytosolic (15 µg) and nuclear extracts (5 µg) from the macrophages used in A was evaluated by Western blot using an anti-p65 Ab (B). Results show a representative experiment out of three. The intensity of the upper band of the binding to the {kappa}B motif in EMSA (mean ± SEM of three experiments, and corresponding to p50.p65 complexes as determined by supershift assays) was analyzed at the indicated times in stimulated macrophages (C).

 
POV and IFN-{gamma} induce tyrosine phosphorylation of I{kappa}B{alpha} and I{kappa}Bß

The preceding results suggest that simultaneous treatment of macrophages with POV and IFN-{gamma} potentiate the activation of NF-{kappa}B. Since NF-{kappa}B activation is dependent on the fate of the inhibitory I{kappa}B proteins, its level and phosphorylation state was determined under these conditions. Stimulation of macrophages with POV for 1 h did not decrease I{kappa}B levels but potentiated the degradation elicited by IFN-{gamma} (Fig. 3GoA). Treatment of cells with POV increased tyrosine phosphorylation of several proteins, among them I{kappa}B{alpha} (25, 26). As Fig. 3GoB shows, incubation of Jurkat cells for 30 min with POV resulted in a decreased electrophoretic mobility of I{kappa}B{alpha}, reflecting a phosphorylation of the protein. However, the bands corresponding to I{kappa}B{alpha} and I{kappa}Bß from macrophages remained unchanged (Fig. 3GoB). In view of these results, we investigated the possibility of I{kappa}B tyrosine phosphorylation as a potential regulatory mechanism governing the fate of the inactive cytosolic NF-{kappa}B complexes. In unstimulated macrophages, tyrosine phosphorylation of I{kappa}B{alpha} or I{kappa}Bß cannot be detected after immunoprecipitation with anti-phosphotyrosine Ab and Western blot analysis with anti-I{kappa}B Abs. However, treatment of macrophages for 15 min with IFN-{gamma}, and especially with POV, resulted in the phosphorylation of these proteins (Fig. 3GoC). Interestingly, the simultaneous presence of both IFN-{gamma} and POV enhanced the amount of phosphorylated I{kappa}B proteins (2-fold increase with respect to cells treated with POV). When the same experiment was analyzed by changing the Abs used for immunoprecipitation and detection of phosphorylated proteins, a similar pattern of protein bands was obtained (Fig. 3GoD). These results confirm the occurrence of tyrosine phosphorylation of I{kappa}B{alpha} and I{kappa}Bß proteins in macrophages treated with POV. However, this modification was not sufficient to activate NF-{kappa}B, but enhanced the degradation of I{kappa}B proteins elicited by IFN-{gamma} and LPS.



View larger version (38K):
[in this window]
[in a new window]
  		FIGURE 3. POV enhances tyrosine phosphorylation of I{kappa}B{alpha} and I{kappa}Bß in macrophages stimulated with IFN-{gamma}. Cultured peritoneal macrophages were stimulated for 1 h with 10 µM POV, 50 U/ml of IFN-{gamma}, 500 ng/ml LPS, or combinations of these. The amount of I{kappa}B{alpha} and I{kappa}Bß proteins was determined in the same blot (A). In a parallel experiment, Jurkat cells and macrophages were stimulated for 30 min as indicated, and the I{kappa}B protein levels were determined in the cytosolic extracts (B). Macrophages incubated for 30 min with 10 µM POV or 50 U/ml of IFN-{gamma} were homogenized and equal amounts of protein from the cytosolic extracts (200 µg) were immunoprecipitated with agarose-conjugated anti-P-tyr Ab (C) or agarose-conjugated anti-I{kappa}B{alpha} and anti-I{kappa}Bß Abs (D). After Western blotting of the immunoprecipitated proteins, the membranes were revealed with anti-I{kappa}B{alpha}/ß or anti-phosphotyrosine Abs, respectively. Results show a representative experiment out of three.

 
POV potentiates IFN-{gamma} signaling in peritoneal macrophages

To determine whether POV might enhance the IFN-{gamma}-dependent signaling involved in iNOS induction, the binding of Stat1{alpha} complexes to a consensus GAS motif was investigated. Treatment of cells with POV was not sufficient to activate the binding to the GAS sequence but significantly potentiates the effect of IFN-{gamma} (not shown). Moreover, the amount of phosphorylated Stat1{alpha} detected by Western blot increased in cells treated with IFN-{gamma}, an effect potentiated notably by POV (Fig. 4Go). In addition to GAS, the iNOS promoter contains two ISREs motifs to which IRF1 binds. Genetic and biochemical analysis of these sequences confirmed their relevance in the transcriptional control of iNOS expression (15, 16, 17, 18, 31). For this reason, the effect of POV on the levels of IRF1 was investigated. As Fig. 4Go shows, the nuclear levels of IRF1 remained unchanged in cells treated with POV. As expected, incubation with IFN-{gamma} increased IRF1 levels, but addition of POV to these cells promoted a rapid and sustained accumulation of IRF1.



View larger version (43K):
[in this window]
[in a new window]
  		FIGURE 4. POV and IFN-{gamma} potentiate Stat1 phosphorylation and IRF1 translocation to the nucleus. Cultured macrophages were treated with 10 µM POV and 50 U/ml of IFN-{gamma} and cell extracts (Stat1) or nuclear extracts (IRF1) were prepared at the indicated times. The levels of Stat1 and IRF1 were determined by Western blot (upper panel). Quantitative analysis of the amount of phosphorylated Stat1 (P-Stat1) and IRF1 was obtained after normalization of the bands corresponding to each time with respect to the content in untreated cells at each time period. Results show the mean of two experiments (lower panel).

 
To gain insight into the transcriptional mechanisms involved in iNOS expression and modulated in response to POV, RAW 264.7 cells (a cell line exhibiting monocyte/macrophage characteristics) (32, 33) were transfected with a plasmid containing a tandem of {kappa}B sequences (({kappa}B)3ConA.CAT) or with plasmids corresponding to sequences constructions of the 5'-flanking region of the iNOS gene (a scheme of the iNOS promoter is shown in Fig. 5GoA). Treatment of cells with POV, IFN-{gamma}, or both did not affect the transfection efficiency when using a common constitutively active expression vector kSV2.CAT (not shown). Incubation of RAW 264.7 with POV was unable to transactivate a construct containing a tandem of three {kappa}B motifs (Fig. 5GoB). However, when these cells were treated with IFN-{gamma} and POV, an important increase in the promoter activity was observed. Analysis of the response of different promoter fragments revealed an important synergism between POV and IFN-{gamma} in terms of activation of a 1.7-kb sequence of the 5'-flanking region of the iNOS gene (p1iNOS.CAT). When cells were transfected with plasmids containing mutated sequences corresponding to the proximal (p2iNOS(+,-).CAT), distal (p2iNOS(-,+).CAT), or both (p2iNOS(-,-).CAT), {kappa}B sites present in the 1.1-kb promoter of iNOS, and stimulated with POV and IFN-{gamma}, a differential effect of each mutation was observed. The promoter mutated in the proximal {kappa}B site exhibited a decreased reporter activity when compared with the native form. However, distal or simultaneous {kappa}B mutations abolished the transcriptional activity of the promoter, reflecting the necessity of the distal {kappa}B site for the expression of the reporter gene in response to POV and IFN-{gamma} (Fig. 5GoB). To better characterize the distal regulatory region, the activity of promoters mutated in the GAS and ISRE sites was analyzed. As Fig. 6Go shows, mutations in the GAS and ISRE motifs decreased the promoter activity in response to IFN-{gamma}; however, in the presence of POV and IFN-{gamma} the reporter activity increased 2- and 2.8-fold for the GAS and ISRE mutants, respectively. These data reflects the relevance of the potentiation by POV of IFN-{gamma}-dependent NF-{kappa}B activation.



View larger version (31K):
[in this window]
[in a new window]
  		FIGURE 5. Analysis of the effect of POV and IFN-{gamma} on the iNOS promoter activity. RAW 264.7 cells were transfected for 6 h with equal amounts of the indicated expression vectors containing a CAT reporter gene (a schematic representation of the iNOS promoter is shown in A). After 24 h of stimulation with the indicated additions, cell extracts were prepared and CAT activity was assayed using the TLC technique. The intensity of the acetylated bands was measured in an autoradiography. Transfection with kSV2.CAT was used to determine the efficiency of the transfection. Results were expressed as the mean band intensity (± SEM; n = 4) after normalization with respect to the activity of untreated cells (B).

 


View larger version (13K):
[in this window]
[in a new window]
  		FIGURE 6. Effect of POV and IFN-{gamma} on the activity of the iNOS promoter mutated in the GAS and ISRE motifs. RAW 264.7 cells were transfected for 6 h with equal amounts of the indicated plasmids containing a luciferase reporter gene. After stimulation with the indicated additions, cell extracts were prepared and luciferase activity was assayed in a luminometer. Results were expressed as the mean ± SEM of the luminescence intensity from four experiments, after normalization with respect to the activity of untreated cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Expression of iNOS in macrophages is achieved through the cooperation between transcription factors present in the promoter region; low doses of IFN-{gamma} or LPS fail to induce the enzyme, but acting in concert, they potentiate the transcription of this gene (12, 13, 14, 15, 16). However, higher concentrations of each are sufficient to trigger the induction of iNOS (15, 31, 34, 35, 36). This would suggest that different signaling pathways convey in the engagement of transcription factors that cooperate in the process. Also, convergent lines of evidence indicate that NF-{kappa}B activation is required for the transcription of the gene (12, 13, 14, 15, 16, 28, 36, 37). Since the ability of IFN-{gamma} to induce iNOS expression in cultured peritoneal macrophages has been observed by several authors (31, 34, 35), we investigated whether NF-{kappa}B could be activated through an IFN-{gamma}-dependent tyrosine phosphorylation of I{kappa}B as described in Jurkat T cells treated with PTP inhibitors (25, 26, 38). Our data show that IFN-{gamma} induces tyrosine phosphorylation of both I{kappa}B{alpha} and I{kappa}Bß, but this is followed by the classic proteolytic degradation of I{kappa}B proteins and translocation of the active NF-{kappa}B complex to the nucleus, although the kinetics of the process is delayed with respect to the response to LPS. The most likely candidate to mediate the IFN-{gamma}-dependent phosphorylation of I{kappa}B is a tyrosine kinase pathway activated by the Jaks associated with the IFN-{gamma} receptor (39, 40). Moreover, when macrophages were treated with POV, an increase of tyrosine phosphorylation of I{kappa}B{alpha} and I{kappa}Bß was detected, but this covalent modification was unable to activate NF-{kappa}B, indicating that in murine macrophages tyrosine phosphorylation is not sufficient to release the I{kappa}B{alpha} or I{kappa}Bß subunits from the complex and to render accessible the nuclear localization signal of NF-{kappa}B. This is despite the close sequence similarities between human and murine I{kappa}B{alpha} and -ß (41, 42, 43). Indeed, using transfection assays with deletion mutants of human I{kappa}B{alpha}, Imbert et al. (25) showed that phosphorylation of tyrosine 42 is responsible for the shift in electrophoretic mobility of I{kappa}B{alpha} and for NF-{kappa}B activation. Interestingly, this residue is conserved perfectly in murine I{kappa}B{alpha} (41). The kinase involved in this phosphorylation in Jurkat cells appears to be Lck, also present in murine macrophages (44), since cells lacking this kinase fail to induce NF-{kappa}B activation after treatment with POV (25). In addition to this, it is conceivable that significant structural differences exist between human and rodent I{kappa}B{alpha} in view of the distinct electrophoretic mobility in SDS-polyacrylamide gels of the phosphorylated I{kappa}B{alpha} from Jurkat cells and from macrophages.

It is also interesting to compare the relative abundance of I{kappa}B{alpha} and -ß in Jurkat cells and macrophages, since the kinetics of degradation and resynthesis of these I{kappa}B proteins, and therefore their biological effects in terms of NF-{kappa}B activation, are quite different: I{kappa}B{alpha} is rapidly degraded and resynthesized because of the presence of {kappa}B motifs in the I{kappa}B{alpha} promoter; however, the degradation of I{kappa}Bß is delayed with respect to I{kappa}B{alpha}, and the resynthesis varies from 1 day to a few hours, depending on the cell type (22, 43, 45). In Jurkat cells, I{kappa}B{alpha} is abundant whereas the amount of I{kappa}Bß is very low; therefore activation of NF-{kappa}B in these cells, either through tyrosine phosphorylation or targeting and degradation, could be explained in terms of I{kappa}B{alpha} turnover. However, the levels of I{kappa}Bß are notably higher in peritoneal macrophages and, therefore, they might contribute to a more sustained NF-{kappa}B activation (22, 43, 45).

Results from various investigators showed that inhibition of tyrosine kinases with inhibitors of broad specificity abolished the expression of iNOS after stimulation with LPS or proinflammatory cytokines (46). However, it appears that redundancy exists in the tyrosine kinase pathways involved in the expression of iNOS since genetic evidence has been obtained indicating that Src family kinases, Hck, Fgr, and Lyn, do not constitute absolute requirements for macrophage activation in response to LPS, IL-1, or TNF-{alpha}, including activation of NF-{kappa}B (47).

Although tyrosine phosphorylation of I{kappa}B{alpha} and -ß is not sufficient to activate NF-{kappa}B in macrophages, it is remarkable that this modification potentiates the well-known pathway of degradation of both I{kappa}B{alpha} and -ß after challenge with IFN-{gamma} or LPS (20, 21). Also, it is possible that phosphorylation of specific tyrosine residues of I{kappa}B could provide the recognition sequence required for the interaction with SH2 domains present in other proteins and, in this way, modulate the fate of the I{kappa}B proteins (25). Since exposure of macrophages and other cells to hypoxia and/or anoxia promotes tyrosine phosphorylation of I{kappa}B{alpha} and the expression of iNOS, it should be expected that this tyrosine phosphorylation of I{kappa}B might contribute to activate NF-{kappa}B, which is a necessary requisite for the expression of iNOS (26, 48, 49). In this regard, it cannot be excluded that the NF-{kappa}B activation observed in Jurkat cells treated with POV might include additional tyrosine phosphorylation of proteins constituting the NF-{kappa}B complex, and with potential contribution to I{kappa}B{alpha} dissociation. In fact, tyrosine phosphorylation of c-Rel has also been described (50).

The mechanism by which POV potentiates iNOS expression in macrophages treated with IFN-{gamma} involves a cooperation between the distal {kappa}B site and GAS or ISRE elements within the iNOS promoter in cells stimulated with IFN-{gamma}, as deduced by transfection experiments. With respect to IRF1 levels, peritoneal macrophages have a basal content of IRF1 that is probably counteracted by the high levels of IRF2 (36). In addition to the increase of IRF1 elicited by IFN-{gamma} and potentiated by POV treatment, the existence of an activation process of IRF1 dependent on tyrosine phosphorylation has also been described (51). Therefore, it should be expected that inhibition of PTP favors tyrosine phosphorylation of IRF1, a process that might contribute to amplifying the response to IFN-{gamma} in the expression of iNOS.

Taken together, the results reported in this work add new possibilities to the control of NF-{kappa}B activation in murine macrophages and reinforce the view of the relevance of synergistic activation between factors involved in host defense and inflammation to generate effective biological responses in these cells (52).


   Acknowledgments
 
We thank Drs. Q.-w. Xie and C. Nathan for the generous gift of the iNOS promoter, Dr. T. J. Evans for the gift of the mutated {kappa}B sequences of the iNOS promoter, and Dr. W. J. Murphy for the gift of the GAS and ISRE mutants. The technical support of O. G. Bodelón and the help of E. Lundin who prepared the manuscript are acknowledged.


   Footnotes
 
1 This work was supported by Grant PM95-0007 from Comisión Interministerial de Ciencia y Technología (Spain). Back

2 Address correspondence and reprint requests to Dr. Lisardo Boscá, Instituto de Bioquímica, Facultad de Farmacia, 28040 Madrid, Spain. E-mail address: Back

3 Abbreviations used in this paper: PTP, protein tyrosine phosphatase; GAS, {gamma}-activated site; iNOS, type II NO synthase; IRF1, IFN regulatory factor I; ISRE, IFN-stimulated response element; I{kappa}B, inhibitor of NF-{kappa}B; POV, peroxovanadate; CAT, chloramphenicol acetyltransferase. Back

Received for publication June 22, 1998. Accepted for publication March 17, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. van Rooijen, N., A. Sanders. 1997. Elimination, blocking, and activation of macrophages: three of a kind?. J. Leukocyte Biol. 62:702.[Abstract]
  2. Celada, A., C. Nathan. 1994. Macrophage activation revisited. Immunol. Today 15:100.[Medline]
  3. Nathan, C.. 1992. Nitric oxide as a secretory product of mammalian cells. FASEB J. 6:3051.[Abstract]
  4. Farrar, M. A., R. D. Schreiber. 1993. The molecular cell biology of IFN-{gamma} and its receptor. Annu. Rev. Immunol. 11:571.[Medline]
  5. Berlett, B. S., E. R. Stadtman. 1997. Protein oxidation in aging, disease, and oxidative stress. J. Biol. Chem. 272:20313.[Free Full Text]
  6. Jr Darnell, J. E., I. M. Kerr, G. R. Stark. 1994. Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science 264:1415.[Abstract/Free Full Text]
  7. Collins, T., M. A. Read, A. S. Neish, M. Z. Whitley, D. Thanos, T. Maniatis. 1995. Transcriptional regulation of endothelial cell adhesion molecules, NF-{kappa}B and cytokine-inducible enhancers. FASEB J. 9:899.[Abstract]
  8. Decker, T., P. Kovarik, A. Meinke. 1997. GAS elements: a few nucleotides with a major impact on cytokine-induced gene expression. J. Interferon Cytokine Res. 17:121.[Medline]
  9. Haque, S. J., V. Flati, A. Deb, B. R. Williams. 1995. Roles of protein-tyrosine phosphatases in Stat1{alpha}-mediated cell signaling. J. Biol. Chem. 270:25709.[Abstract/Free Full Text]
  10. Ruff, S. J., K. Chen, S. Cohen. 1997. Peroxovanadate induces tyrosine phosphorylation of multiple signaling proteins in mouse liver and kidney. J. Biol. Chem. 272:1263.[Abstract/Free Full Text]
  11. Haque, S. J., Q. Wu, W. Kammer, K. Friedrich, J. M. Smith, I. M. Kerr, G. R. Stark, B. R. Williams. 1997. Receptor-associated constitutive protein tyrosine phosphatase activity controls the kinase function of JAK1. Proc. Natl. Acad. Sci. USA 94:8563.[Abstract/Free Full Text]
  12. Nathan, C., Q.-w. Xie. 1994. Nitric oxide synthases: roles, tolls, and controls. Cell 78:915.[Medline]
  13. Nathan, C., Q.-w. Xie. 1994. Regulation of biosynthesis of nitric oxide. J. Biol. Chem. 269:13725.[Free Full Text]
  14. MacMicking, J., Q.-w. Xie, C. Nathan. 1997. Nitric oxide and macrophage function. Annu. Rev. Immunol. 15:323.[Medline]
  15. Gao, J., D. C. Morrison, T. J. Parmely, S. W. Russell, W. J. Murphy. 1997. An IFN-{gamma}-activated site (GAS) is necessary for full expression of the mouse iNOS gene in response to IFN-{gamma} and lipopolysaccharide. J. Biol. Chem. 272:1226.[Abstract/Free Full Text]
  16. Martin, E., C. Nathan, Q.-w. Xie. 1994. Role of interferon regulatory factor 1 in induction of nitric oxide synthase. J. Exp. Med. 180:977.[Abstract/Free Full Text]
  17. Lowenstein, C. J., E. W. Alley, P. Raval, A. M. Snowman, S. H. Snyder, S. W. Russell, W. J. Murphy. 1993. Macrophage nitric oxide synthase gene: two upstream regions mediate induction by IFN-{gamma} and lipopolysaccharide. Proc. Natl. Acad. Sci. USA 90:9730.[Abstract/Free Full Text]
  18. Xie, Q.-w., R. Whisnant, C. Nathan. 1993. Promoter of the mouse gene encoding calcium-independent nitric oxide synthase confers inducibility by interferon-{gamma} and bacterial lipopolysaccharide. J. Exp. Med. 177:1779.[Abstract/Free Full Text]
  19. Goldring, C. E., S. Reveneau, M. Algarte, J. F. Jeannin. 1996. In vivo footprinting of the mouse inducible nitric oxide synthase gene: inducible protein occupation of numerous sites including Oct and NF-IL6. Nucleic Acids Res. 24:1682.[Abstract/Free Full Text]
  20. Baeuerle, P. A., T. Henkel. 1994. Function and activation of NF-{kappa}B in the immune system. Annu. Rev. Immunol. 12:141.[Medline]
  21. Thanos, D., T. Maniatis. 1995. NF-{kappa}B: a lesson in family values. Cell 80:529.[Medline]
  22. Velasco, M., M. J. M. Diaz-Guerra, P. Martin-Sanz, A. Alvarez, L. Boscá. 1997. Rapid up-regulation of I{kappa}Bß and abrogation of NF-{kappa}B activity in peritoneal macrophages stimulated with lipopolysaccharide. J. Biol. Chem. 272:23025.[Abstract/Free Full Text]
  23. Woronicz, J. D., X. Gao, Z. Cao, M. Rothe, D. V. Goeddel. 1997. I{kappa}B kinase-{kappa}: NF-{kappa}B activation and complex formation with I{kappa}B kinase-{alpha} and NIK. Science 278:866.[Abstract/Free Full Text]
  24. Mercurio, F., H. Zhu, B. W. Murray, A. Shevchenko, B. L. Bennett, J. Li, D. B. Young, M. Barbosa, M. Mann, A. Manning, A. Rao. 1997. IKK-1 and IKK-2: cytokine activated I{kappa}B kinases essential for NF-{kappa}B activation. Science 278:860.[Abstract/Free Full Text]
  25. Imbert, V., R. A. Rupec, A. Livolsi, H. L. Pahl, E. B. Traenckner, C. Mueller-Dieckmann, D. Farahifar, B. Rossi, P. Auberger, P. A. Baeuerle. 1996. Tyrosine phosphorylation of I{kappa}B-{alpha} activates NF{kappa}B without proteolytic degradation of I{kappa}B-{alpha}. Cell 86:787.[Medline]
  26. Koong, A. C., E. Y. Chen, N. F. Mivechi, N. C. Denko, P. Stambrook, A. J. Giaccia. 1994. Hypoxic activation of nuclear factor-{kappa}B is mediated by a Ras and Raf signaling pathway and does not involve MAP kinase (ERK1 or ERK2). Cancer Res. 54:5273.[Abstract/Free Full Text]
  27. Diaz-Guerra, M. J. M., M. Velasco, P. Martin-Sanz, L. Bosca. 1996. Evidence for common mechanisms in the transcriptional control of type II nitric oxide synthase in isolated hepatocytes: requirement of NF-{kappa}B activation after stimulation with bacterial cell wall products and phorbol esters. J. Biol. Chem. 271:30114.[Abstract/Free Full Text]
  28. Spink, J., J. Cohen, T. J. Evans. 1995. The cytokine responsive vascular smooth muscle cell enhancer of inducible nitric oxide synthase: activation by nuclear factor-{kappa}B. J. Biol. Chem. 270:29541.[Abstract/Free Full Text]
  29. Schreiber, E., P. Matthias, M. M. Muller, W. Schaffner. 1989. Rapid detection of octamer binding proteins with "mini-extracts" prepared from a small number of cells. Nucleic Acids Res. 17:6419.[Free Full Text]
  30. Sadowski, H. B., K. Shuai, Jr J. E. Darnell, M. Z. Gilman. 1993. A common nuclear signal transduction pathway activated by growth factor and cytokine receptors. Science 261:1739.[Abstract/Free Full Text]
  31. Kamijo, R., H. Harada, T. Matsuyama, M. Bosland, J. Gerecitano, D. Shapiro, J. Le, S. I. Koh, T. Kimura, S. J. Green, T. W. Mak, T. Taniguchi, J. Vilcek. 1994. Requirement for transcription factor IRF-1 in NO synthase induction in macrophages. Science 263:1612.[Abstract/Free Full Text]
  32. Ralph, P., I. Nakoinz. 1977. Antibody-dependent killing of erythrocyte and tumor targets by macrophage-related cell lines: enrichment by PPD and LPS. J. Immunol. 119:950.[Abstract/Free Full Text]
  33. Raschke, W. C., S. Baird, P. Ralph, I. Nakoinz. 1978. Functional macrophage cell lines transformed by Abelson leukemia virus. Cell 15:261.[Medline]
  34. Lorsbach, R. B., W. J. Murphy, C. J. Lowenstein, S. H. Snyder, S. W. Russell. 1993. Expression of the nitric oxide synthase gene in mouse macrophages activated for tumor cell killing: molecular basis for the synergy between interferon-{gamma} and lipopolysaccharide. J. Biol. Chem. 268:1908.[Abstract/Free Full Text]
  35. Ricote, M., A. C. Li, T. M. Willson, C. J. Kelly, C. K. Glass. 1998. The peroxisome proliferator-activated receptor-{gamma} is a negative regulator of macrophage activation. Nature 391:79.[Medline]
  36. Lopez-Collazo, E., S. Hortelano, A. Rojas, L. Bosca. 1998. Triggering of peritoneal macrophages with IFN-{alpha}/ß attenuates the expression of inducible nitric oxide synthase through a decrease in NF-{kappa}B activation. J. Immunol. 160:2889.[Abstract/Free Full Text]
  37. Griscavage, J. M., S. Wilk, L. J. Ignarro. 1996. Inhibitors of the proteasome pathway interfere with induction of nitric oxide synthase in macrophages by blocking activation of transcription factor NF-{kappa}B. Proc. Natl. Acad. Sci. USA 93:3308.[Abstract/Free Full Text]
  38. Singh, S., B. G. Darnay, B. B. Aggarwal. 1996. Site-specific tyrosine phosphorylation of I{kappa}B{alpha} negatively regulates its inducible phosphorylation and degradation. J. Biol. Chem. 271:31049.[Abstract/Free Full Text]
  39. Weiss, A., J. Schlessinger. 1998. Switching signals on or off by receptor dimerization. Cell 94:277.[Medline]
  40. English, B. K., S. L. Orlicek, Z. Mei, E. A. Meals. 1997. Bacterial LPS and IFN-{gamma} trigger the tyrosine phosphorylation of vav in macrophages: evidence for involvement of the hck tyrosine kinase. J. Leukocyte Biol. 62:859.[Abstract]
  41. Noble, P. W., C. M. McKee, M. Cowman, H. S. Shin. 1996. Hyaluronan fragments activate an NF-{kappa}B/I-{kappa}B{alpha} autoregulatory loop in murine macrophages. J. Exp. Med. 183:2373.[Abstract/Free Full Text]
  42. Sonoda, Y., T. Kasahara, Y. Yamaguchi, K. Kuno, K. Matsushima, N. Mukaida. 1997. Stimulation of interleukin-8 production by okadaic acid and vanadate in a human promyelocyte cell line, an HL-60 subline: possible role of mitogen-activated protein kinase on the okadaic acid-induced NF-{kappa}B activation. J. Biol. Chem. 272:15366.[Abstract/Free Full Text]
  43. Thompson, J. E., R. J. Phillips, H. Erdjument-Bromage, P. Tempst, S. Ghosh. 1995. I{kappa}B-ß regulates the persistent response in a biphasic activation of NF-{kappa}B. Cell 80:573.[Medline]
  44. Ishii, T., T. Yanagawa, T. Kawane, K. Yuki, J. Seita, H. Yoshida, S. Bannai. 1996. Murine peritoneal macrophages induce a novel 60-kDa protein with structural similarity to a tyrosine kinase p56lck-associated protein in response to oxidative stress. Biochem. Biophys. Res. Commun. 226:456.[Medline]
  45. Stancovski, I., D. Baltimore. 1997. NF-{kappa}B activation: the I{kappa}B kinase revealed?. Cell 91:299.[Medline]
  46. Ruetten, H., C. Thiemermann. 1997. Effects of tyrphostins and genistein on the circulatory failure and organ dysfunction caused by endotoxin in the rat: a possible role for protein tyrosine kinase. Br. J. Pharmacol. 122:59.[Medline]
  47. Meng, F., C. A. Lowell. 1997. Lipopolysaccharide (LPS)-induced macrophage activation and signal transduction in the absence of Src-family kinases Hck, Fgr, and Lyn. J. Exp. Med. 185:1661.[Abstract/Free Full Text]
  48. Melillo, G., L. S. Taylor, A. Brooks, G. W. Cox, L. Varesio. 1996. Regulation of inducible nitric oxide synthase expression in IFN-{gamma}-treated murine macrophages cultured under hypoxic conditions. J. Immunol. 157:2638.[Abstract]
  49. Melillo, G., L. S. Taylor, A. Brooks, T. Musso, G. W. Cox, L. Varesio. 1997. Functional requirement of the hypoxia-responsive element in the activation of the inducible nitric oxide synthase promoter by the iron chelator desferrioxamine. J. Biol. Chem. 272:12236.[Abstract/Free Full Text]
  50. Druker, B. J., M. Neumann, K. Okuda, Jr B. R. Franza, J. D. Griffin. 1994. rel Is rapidly tyrosine-phosphorylated following granulocyte-colony stimulating factor treatment of human neutrophils. J. Biol. Chem. 269:5387.[Abstract/Free Full Text]
  51. Sharf, R., D. Meraro, A. Azriel, A. M. Thornton, K. Ozato, E. F. Petricoin, A. C. Larner, F. Schaper, H. Hauser, B. Z. Levi. 1997. Phosphorylation events modulate the ability of interferon consensus sequence binding protein to interact with interferon regulatory factors and to bind DNA. J. Biol. Chem. 272:9785.[Abstract/Free Full Text]
  52. Cheschire, J. L., Jr A. S. Baldwin. 1997. Synergistic activation of NF-{kappa}B by tumor necrosis factor {alpha} and {gamma} interferon via enhanced I{kappa}B{alpha} degradation and de novo I{kappa}Bß degradation. Mol. Cell. Biol. 17:6746.[Abstract]



This article has been cited by other articles:


Home page
 Am. J. Pathol.Home page
J. Cuenca, N. Goren, P. Prieto, P. Martin-Sanz, and L. Bosca
Selective Impairment of Nuclear Factor-{kappa}B-Dependent Gene Transcription in Adult Cardiomyocytes: Relevance for the Regulation of the Inflammatory Response in the Heart
Am. J. Pathol., September 1, 2007; 171(3): 820 - 828.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
J. Blanchette, P. Pouliot, and M. Olivier
Role of protein tyrosine phosphatases in the regulation of interferon-{gamma}-induced macrophage nitric oxide generation: implication of ERK pathway and AP-1 activation
J. Leukoc. Biol., March 1, 2007; 81(3): 835 - 844.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
V. Vila-del Sol, M. D. Diaz-Munoz, and M. Fresno
Requirement of tumor necrosis factor {alpha} and nuclear factor-{kappa}B in the induction by IFN-{gamma} of inducible nitric oxide synthase in macrophages
J. Leukoc. Biol., January 1, 2007; 81(1): 272 - 283.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
G. Forget, D. J. Gregory, L. A. Whitcombe, and M. Olivier
Role of Host Protein Tyrosine Phosphatase SHP-1 in Leishmania donovani-Induced Inhibition of Nitric Oxide Production.
Infect. Immun., November 1, 2006; 74(11): 6272 - 6279.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
M. Brown, M. McGuinness, T. Wright, X. Ren, Y. Wang, G. P. Boivin, H. Hahn, A. M. Feldman, and W. K. Jones
Cardiac-specific blockade of NF-{kappa}B in cardiac pathophysiology: differences between acute and chronic stimuli in vivo
Am J Physiol Heart Circ Physiol, July 1, 2005; 289(1): H466 - H476.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
V. Vila-del Sol and M. Fresno
Involvement of TNF and NF-{kappa}B in the Transcriptional Control of Cyclooxygenase-2 Expression by IFN-{gamma} in Macrophages
J. Immunol., March 1, 2005; 174(5): 2825 - 2833.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
C.-W. Chen, Y.-H. Chang, C.-J. Tsi, and W.-W. Lin
Inhibition of IFN-{gamma}-Mediated Inducible Nitric Oxide Synthase Induction by the Peroxisome Proliferator-Activated Receptor {gamma} Agonist, 15-Deoxy-{Delta}12,14-Prostaglandin J2, Involves Inhibition of the Upstream Janus Kinase/STAT1 Signaling Pathway
J. Immunol., July 15, 2003; 171(2): 979 - 988.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
N. A. Callejas, A. Fernandez-Martinez, A. Castrillo, L. Bosca, and P. Martin-Sanz
Selective Inhibitors of Cyclooxygenase-2 Delay the Activation of Nuclear Factor kappa B and Attenuate the Expression of Inflammatory Genes in Murine Macrophages Treated with Lipopolysaccharide
Mol. Pharmacol., March 1, 2003; 63(3): 671 - 677.
[Abstract] [Full Text] [PDF]

Home page
 JEMHome page
A. Castrillo, D. J. Pennington, F. Otto, P. J. Parker, M. J. Owen, and L. Bosca
Protein Kinase C{epsilon} Is Required for Macrophage Activation and Defense Against Bacterial Infection
J. Exp. Med., October 29, 2001; 194(9): 1231 - 1242.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
A. Aicher, M. Hayden-Ledbetter, W. A. Brady, A. Pezzutto, G. Richter, D. Magaletti, S. Buckwalter, J. A. Ledbetter, and E. A. Clark
Characterization of Human Inducible Costimulator Ligand Expression and Function
J. Immunol., May 1, 2000; 164(9): 4689 - 4696.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
A. Castrillo, M. J. M. Díaz-Guerra, S. Hortelano, P. Martín-Sanz, and L. Boscá
Inhibition of Ikappa B Kinase and Ikappa B Phosphorylation by 15-Deoxy-Delta 12,14-Prostaglandin J2 in Activated Murine Macrophages
Mol. Cell. Biol., March 1, 2000; 20(5): 1692 - 1698.
[Abstract] [Full Text]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Díaz-Guerra, M. J. M.
Right arrow Articles by Boscá, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Díaz-Guerra, M. J. M.
Right arrow Articles by Boscá, L.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS