IFN-gamma  + LPS induction of iNOS is modulated by ERK, JNK/SAPK, and p38mapk in a mouse macrophage cell line

Edward D. Chan1 and David W. H. Riches2

1 Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado Health Sciences Center, and 2 Program in Cell Biology, National Jewish Medical and Research Center, Denver, Colorado 80206


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Nitric oxide (NO·) produced by inducible nitric oxide synthase (iNOS) mediates a number of important physiological and pathophysiological processes. The objective of this investigation was to examine the role of mitogen-activated protein kinases (MAPKs) in the regulation of iNOS and NO· by interferon-gamma (IFN-gamma ) + lipopolysaccharide (LPS) in macrophages using specific inhibitors and dominant inhibitory mutant proteins of the MAPK pathways. The signaling pathway utilized by IFN-gamma in iNOS induction is well elucidated. To study signaling pathways that are restricted to the LPS-signaling arm, we used a subclone of the parental RAW 264.7 cell line that is unresponsive to IFN-gamma alone with respect to iNOS induction. In this RAW 264.7gamma NO(-) subclone, IFN-gamma and LPS are nevertheless required for synergistic activation of the iNOS promoter. We found that extracellular signal-regulated kinase (ERK) augmented and p38mapk inhibited IFN-gamma  + LPS induction of iNOS. Dominant-negative MAPK kinase-4 inhibited iNOS promoter activation by IFN-gamma  + LPS, also implicating the c-Jun NH2-terminal kinase (JNK) pathway in mediating iNOS induction. Inhibition of the ERK pathway markedly reduced IFN-gamma  + LPS-induced tumor necrosis factor-alpha protein expression, providing a possible mechanism by which ERK augments iNOS expression. The inhibitory effect of p38mapk appears more complex and may be due to the ability of p38mapk to inhibit LPS-induced JNK activation. These results indicate that the MAPKs are important regulators of iNOS-NO· expression by IFN-gamma  + LPS.

nitric oxide; monocytes/macrophages; protein kinases; lipopolysaccharide; rodent


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

NITRIC OXIDE (NO·) is a free radical produced from the catalytic oxidation of the guanidino group of L-arginine by nitric oxide synthase (NOS). NO· has been shown to have a number of important biological functions, including tumor cell killing, host defense against intracellular pathogens, vasodilatation, neurotransmission, and inhibition of platelet aggregation (29). NO· produced from the constitutive forms of NOS, namely, endothelial and neuronal NOS, functions principally as a vasodilator and neurotransmitter, respectively (41). The third form of NOS, known as inducible NOS (iNOS or NOS II), is generally not present in resting cells but is induced by various stimuli, which include bacterial lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNF-alpha ), interleukin (IL)-1beta , picolinic acid, lipoarabinomannan, phorbol ester, interferon (IFN)-gamma , and hypoxia (11, 35, 36, 40). In contrast to the constitutive forms of NOS, iNOS is regulated primarily at the level of transcription, because calmodulin is already tightly bound and, thus, iNOS expression is largely independent of intracellular calcium (33, 40). Increased expression of iNOS has been associated with disorders as diverse as septic and hemorrhagic shock, rheumatoid arthritis, and chronic infections such as tuberculosis.

Since the cloning of the mouse iNOS gene, the transcriptional regulation of iNOS has been extensively characterized (30, 31, 55, 56). Although many potential cis-regulatory elements have been identified, six enhancer elements have been shown to be important in IFN-gamma  + LPS induction of iNOS (15, 17, 24, 34, 39, 57). In the upstream enhancer element known as region II, located between positions -913 and -1029, two IFN-stimulated response elements and an IFN-gamma -activated sequence have been identified to be critical for IFN-gamma signaling of iNOS transcription (15, 24, 34). For LPS transcriptional regulation of iNOS, two nuclear factor-kappa B (NF-kappa B) sites, one located in the basal enhancer region I between positions -48 and -209 and another located in region II, are required (16, 39, 55, 57). More recently, another cis-regulatory element, an Oct site, which binds the basal transcriptional element octomer, was found to be required for maximal iNOS transcription by LPS (15, 17). NF-kappa B has also been shown to be required in the regulation of the human iNOS gene (49).

The mitogen-activated protein kinases (MAPKs) are Ser-Thr kinases that have been shown to activate a number of transcription factors, including activator protein-1 (AP-1), activating transcription factor (ATF)-2, cAMP-responsive element binding protein, NF-kappa B, and certain members of the Ets family (19, 22, 37). MAPKs are comprised of three principal family members with distinct isoforms within each member: extracellular signal-regulated kinases (ERKs), p38mapks, and c-Jun NH2-terminal kinases (JNKs) or stress-activated protein kinases (SAPKs) (9, 20, 21, 26). MAPKs are activated by a family of dual-specificity kinases [MAPK kinases (MKKs)] that phosphorylate MAPKs on specific Thr and Tyr residues: MKK4 and MKK7 [also known as JNKK1 and JNKK2 or SAPK/ERK kinase (SEK)-1 and -2] activate the JNK/SAPKs, MKK3 and MKK6 activate the p38mapks, and MAPK/ERK kinase (MEK)-1 and -2 activate the ERKs (10, 28, 45). Further upstream, the MEK kinases (MEKKs) and other kinases such as germinal center kinase, mixed-lineage kinase, p21-activated kinase, and tyrosine phenol-lyase are known to activate the JNK and ERK pathways (8, 13, 42). We and others previously examined the role of the MAPKs in the induction of iNOS by TNF-alpha and other stimuli (2, 3, 5, 7, 18, 43, 51). We showed that the MEKK1-MKK4-JNK pathway played an important role in the transcriptional regulation of iNOS by TNF-alpha (5). Furthermore, we showed that ERK and p38mapk were not involved in the regulation of iNOS by IFN-gamma  + TNF-alpha . We undertook this study to determine the role of the MAPKs in the regulation of iNOS with IFN-gamma  + LPS stimulation (Fig. 1). Because the IFN-gamma -signaling pathway has been elucidated in regard to iNOS induction, we restricted our study to the MAPK family of signaling molecules that is activated by LPS. The parental RAW 264.7 macrophage strain is known to express iNOS-NO· in response to IFN-gamma alone, making it difficult to discriminate the relative contribution of IFN-gamma and LPS on costimulation. Moreover, the use of cells that are capable of producing NO· with IFN-gamma stimulation alone would be problematic when pharmacological or dominant-negative (DN) inhibitors of the LPS-induced MAPK pathways are utilized to sort out the regulatory signaling pathways. To eliminate this obstacle, we used a subclone of RAW 264.7 cells that does not respond to IFN-gamma alone with respect to iNOS induction (39). Nevertheless, in these RAW 264.7gamma NO(-) cells, it is important to emphasize that costimulation with IFN-gamma and LPS is essential for induction of iNOS-NO· (39).


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Fig. 1.   Simplified diagram of the hypothesized mitogen-activated protein kinase (MAPK) signaling pathway(s) in lipopolysaccharide (LPS) induction of inducible nitric oxide (NO) synthase (iNOS)-NO·. The elucidated and necessary interferon-gamma (IFN-gamma )-signaling arm is also noted. ERK, extracellular signal-regulated kinase; MEK, MAPK/ERK kinase; MKK, MAPK kinase; JNK, c-Jun NH2-terminal kinase; MEKK, MEK kinase; +ve, positive.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Materials. RAW 264.7gamma NO(-) macrophages, which were used for all the studies, were generously provided by Dr. William J. Murphy (University of Kansas Medical Center) (39). Unlike the parent RAW 264.7 line (TIB-71; American Type Culture Collection, Rockville, MD), the RAW 264.7gamma NO(-) line does not respond to IFN-gamma alone by producing NO·. LPS purified from Salmonella typhimurium was purchased from Sigma Chemical (St. Louis, MO). Fetal bovine serum (FBS) was purchased from Irvine Scientific (Irvine, CA). Glutathione-Sepharose beads were purchased from Pharmacia (Piscataway, NJ). Enhanced chemiluminescence assay kits were obtained from Amersham Life Sciences (Arlington Heights, IL). Recombinant c-Jun-(1-79)-glutathione S-transferase (GST) and DN-MEKK1 mutant in a cDNA3 expression vector were kindly provided by Dr. Gary Johnson (University of Colorado Health Sciences Center). The DN-MKK4 mutant (K116R) and DN-c-Jun, which lacks the transactivating domain but contains the DNA-binding domain (DBD-c-Jun), both in an LNCX expression vector, were gifts from Dr. Lynn Heasley (University of Colorado Health Sciences Center). Rabbit polyclonal anti-p46 JNK, rabbit polyclonal anti-p42 ERK, and mouse monoclonal phospho-specific p46-p54 JNK antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse IFN-gamma and neutralizing anti-TNF-alpha and anti-IL-1beta antibodies were obtained from R & D Systems (Minneapolis, MN). Rabbit anti-iNOS polyclonal antibody was purchased from Alexis (San Diego, CA). [gamma -32P]ATP (>3,000 Ci/mmol) was purchased from NEN Research Products DuPont (Wilmington, DE). The MEK1 inhibitor (PD-98059) and phospho-specific p38mapk antibody were purchased from New England Biolabs (Beverly, MA). SB-203580 (p38mapk inhibitor) was purchased from Calbiochem (San Diego, CA). The iNOS-luciferase reporter construct was generously provided by Dr. Charles J. Lowenstein (Johns Hopkins University School of Medicine, Baltimore, MD) and Dr. Robert Scheinman (University of Colorado Health Sciences Center). Phospho-specific ERK polyclonal antibody and the firefly luciferase reporter assay system were purchased from Promega (Madison, WI). The Lipofectamine reagent used for the transfection experiments was purchased from GIBCO BRL (Gaithersburg, MD). TNF-alpha ELISA kit was purchased from Genzyme (Cambridge, MA). The cytomegalovirus beta -galactosidase plasmid used for normalizing transfection efficiency was kindly provided by Dr. Dwight Klemm (National Jewish Medical and Research Center). All other reagents were of the highest purity.

Analysis of nitrite anion accumulation. Nitrite anion (NO<SUB>2</SUB><SUP>−</SUP>) accumulation in the supernatant was determined as previously reported (11). Briefly, RAW 264.7gamma NO(-) cell monolayers were stimulated with LPS (1 ng/ml) and IFN-gamma (10 U/ml) or were coincubated with PD-98059 (30 µM) or SB-203580 (30 µM) for 18 h. One hundred microliters of supernatant were combined with an equal volume of Greiss reagent, and the samples were incubated at room temperature for 10 min before the absorbance was quantified at 550 nm. With the use of a standard curve, the nanomoles of NO<SUB>2</SUB><SUP>−</SUP> produced were determined and normalized to total cell number in each sample.

Determination of JNK activity. For measurement of JNK activity, the RAW 264.7gamma NO(-) cells were lysed at 4°C with 500 µl of ice-cold lysis buffer [50 mM Tris · HCl, pH 8.0, containing 137 mM NaCl, 10% (vol/vol) glycerol, 1% (vol/vol) Nonidet P-40, 1 mM NaF, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 2 mM Na3VO4, and 1 mM phenylmethylsulfonyl fluoride (21)]. After the protein content was normalized between samples, JNK in each sample of lysate was bound to 15 µl of a 1:1 slurry of lysis buffer-GST-c-Jun-(1-79)-Sepharose beads and incubated at 4°C for 2 h. The beads were then washed twice with 500 µl of lysis buffer and twice with 500 µl of JNK buffer (20 mM HEPES buffer, pH 7.2, containing 30 mM beta -glycerophosphate, 10 mM p-nitrophenylphosphate, 10 mM MgCl2, 0.5 mM dithiothreitol, and 50 µM Na3VO4). The activity of JNK was detected by phosphorylation of c-Jun-GST in an in vitro kinase assay and was assessed by incorporation of [gamma -32P]ATP (10 µCi/sample) in JNK buffer incubated at 30°C for 30 min. The kinase reactions were then stopped with an equal volume of 2× Laemmli sample buffer containing 20 mM dithiothreitol and boiled for 3 min. The proteins present in the supernatants were separated by SDS-PAGE through a 12% polyacrylamide gel and transferred onto nitrocellulose membranes. 32P-labeled c-Jun-GST was detected by autoradiography.

Western blot analysis. Samples were separated by SDS-PAGE and transferred onto nitrocellulose membranes as described elsewhere (50). The blots were then washed in Tris-Tween-buffered saline [TTBS, 20 mM Tris · HCl buffer, pH 7.6, containing 137 mM NaCl and 0.05% (vol/vol) Tween 20], blocked overnight with 5% (wt/vol) nonfat dry milk, and probed according to the method described by Towbin et al. (50) with a polyclonal iNOS antibody or with phospho-specific antibodies to p46-p54 JNK, p42/p44 ERK, and p38mapk antibodies in 5% (wt/vol) BSA dissolved in TTBS. With the use of horseradish peroxidase-conjugated secondary anti-rabbit or anti-mouse antibody, bound antibodies were detected by enhanced chemiluminescence. To determine equal loading of proteins between samples, the membranes were probed with rabbit polyclonal p46 JNK, p42/p44 ERK, and p38mapk antibodies.

Transient transfection and luciferase assay. RAW 264.7gamma NO(-) cells were plated at a density of 1 × 106 cells per six-well plate in RPMI containing penicillin (100 U/ml), streptomycin (100 µg/ml), and 10% (vol/vol) heat-inactivated FBS. After 24 h of growth to ~30-40% confluence, the cells were transfected with plasmids using Lipofectamine as described by the manufacturer's protocol (GIBCO BRL). Briefly, 0.3 µg of iNOS-luceriferase plasmid was combined with 2 µg of DN-MEKK, 2 µg of DN-MKK4, or 2 µg of DBD-c-Jun plasmid, 10 µl of Lipofectamine reagent, and 100 µl of Optimem serum-free medium. To normalize for the amount of DNA transfected, equivalent amounts of the pc-DNA3 empty vector (for DN-MEKK1) or LNCX empty vector (for DN-MKK4 or DBD-c-Jun) were cotransfected in the controls. To normalize for transfection efficiency between samples, 1 µg of cytomegalovirus beta -galactosidase plasmid was cotransfected for each sample. The lipid-DNA mixture was incubated for 30 min at room temperature. Each well was then washed with 2 ml of Optimem medium and replaced with 1 ml of the Lipofectamine-DNA mixture. After 5 h of incubation, 1 ml of RPMI containing 20% (vol/vol) FBS and 1% penicillin-streptomycin-L-glutamine was added to each well. The media were changed 24 h after transfection, and after an additional 48 h, the cells were stimulated with IFN-gamma (10 U/ml) and LPS (1 ng/ml) for 8 h. The cells were then washed with PBS, lysed in a luciferase lysis buffer, and assayed for luciferase activity according to the manufacturer's instructions. The amount of luciferase activity was normalized to beta -galactosidase activity and reported as magnitude increase in activity.

Statistical analysis. Replicate experiments were independent, and summary results are presented as means ± SE. Differences were considered significant for P < 0.05. Group means were compared by repeated-measures ANOVA using Fisher's least significant difference.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

IFN-gamma synergizes with LPS in the expression of NO<SUB>2</SUB><SUP>−</SUP>. We first showed that, unlike the parental RAW 264.7 cell line available from American Type Culture Collection, these selected RAW 264.7gamma NO(-) cells do not produce NO· with IFN-gamma stimulation alone. We considered it paramount to use this macrophage phenotype, because it would allow the detection of any effects of inhibitors of the LPS-induced MAPK signaling pathways on iNOS-NO· induction. Thus RAW 264.7gamma NO(-) macrophages were stimulated with IFN-gamma (10 U/ml), LPS (1 ng/ml), or both for 18 h, and NO<SUB>2</SUB><SUP>−</SUP> (a stable metabolite of NO·) levels in the culture supernatants were measured using the Greiss reagent assay. As shown in Fig. 2, cells stimulated with IFN-gamma or LPS produced no more NO<SUB>2</SUB><SUP>−</SUP> than unstimulated cells. In contrast, there was marked synergy of NO<SUB>2</SUB><SUP>−</SUP> production when cells were costimulated with IFN-gamma  + LPS.


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Fig. 2.   RAW 264.7gamma NO(-) cells do not respond to IFN-gamma alone in NO· expression. Macrophages were stimulated with IFN-gamma (10 U/ml), LPS (1 ng/ml), or both for 18 h and then subjected to Greiss reagent assay for NO<SUB>2</SUB><SUP>−</SUP> accumulation in the supernatant. Results are means of 3 independent experiments. ***P < 0.001 compared with all other treatments.

LPS induces phosphorylation of ERK, p38mapk, and JNK. Activation of the MAPKs is dependent on the phosphorylation by their respective upstream MAPK kinases of Thr and Tyr residues on MAPK-specific tripeptide motifs: TPY for JNK, TEY for ERK, and TGY for p38mapk. Thus, before the study of the role of the MAPKs in the regulation of iNOS by LPS, we examined the phosphorylation of the MAPKs by LPS using phospho-specific antibodies that recognize the TXY motifs. RAW 264.7gamma NO(-) macrophages were stimulated with LPS at 100 ng/ml for 0-3 h. The cells were then lysed with Nonidet P-40 lysis buffer, and the whole cell lysates were normalized for protein content and then separated by SDS-PAGE. This was followed by immunoblot of the separated proteins with phospho-specific antibodies to each of the MAPKs. As shown in Fig. 3, phosphorylation of Thr and Tyr residues of all three MAPKs occurred after 30 min of LPS stimulation, peaking at 60 min, and remained sustained above basal level even after 3 h of stimulation. This pattern of phosphorylation is significantly more prolonged than that induced by TNF-alpha stimulation in bone marrow-derived macrophages, in which the MAPKs were phosphorylated and activated after only 10 min of stimulation and returned to near-basal level by 30 min (4, 53, 54). IFN-gamma alone does not activate any of the MAPKs (0-18 h of stimulation), and costimulation of IFN-gamma  + LPS does not alter the kinetics of MAPK activation compared with LPS alone (data not shown).


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Fig. 3.   Phosphorylation of TXY motifs of ERK (A), p38mapk (B), and JNK (C) with LPS stimulation. RAW 264.7gamma NO(-) cells were stimulated with LPS (100 ng/ml) for 0-180 min, and then nuclear-free whole cell lysates were blotted with phospho-specific antibodies to each of the MAPKs. Result is representative of 2 independent experiments.

MEK1-ERK inhibition attenuates NO<SUB>2</SUB><SUP>−</SUP> expression by inhibition of LPS-induced TNF-alpha production. To investigate the role of ERK in the modulation of iNOS expression by LPS, we determined the effect of PD-98059, a specific inhibitor of MEK1-ERK, on NO<SUB>2</SUB><SUP>−</SUP> production. Macrophages were pretreated with PD-98059 (30 µM) for 1 h and then costimulated with IFN-gamma  + LPS in the presence of the inhibitor for 18 h before quantification of NO<SUB>2</SUB><SUP>−</SUP> accumulation in culture supernatants. Compared with IFN-gamma  + LPS stimulation alone, treatment with PD-98059 reduced NO<SUB>2</SUB><SUP>−</SUP> production by ~25-30% (Fig. 4A), suggesting that ERK plays a modest, although positive, regulatory role in the induction of iNOS by IFN-gamma  + LPS. The vehicle DMSO, at a concentration equivalent to 30 µM PD-98059 (0.075%), did not affect stimulated NO<SUB>2</SUB><SUP>−</SUP> expression. To examine whether iNOS protein level was also influenced by PD-98059, we treated the RAW 264.7gamma NO(-) cells as described above and, after lysis with a Nonidet P-40 buffer, performed an immunoblot on nuclear-free whole cell lysate with a polyclonal iNOS antibody. As shown in Fig. 4B, PD-98059 also inhibited iNOS protein expression in a fashion that was similar to that observed for NO<SUB>2</SUB><SUP>−</SUP>, whereas DMSO had no effect. A possible toxic or antiproliferative effect of PD-98059 was assessed by a cell viability assay in which metabolically induced formazan production was measured (CellTiter 96 Aqueous One Solution Cell Proliferation Assay; Promega). Compared with unstimulated cells or cells stimulated with IFN-gamma  + LPS, addition of 30 µM PD-98059 for 18 h had no effect on cell viability (data not shown).


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Fig. 4.   A: effects of PD-98059 and SB-203580 on IFN-gamma  + LPS-induced NO<SUB>2</SUB><SUP>−</SUP> expression. RAW 264.7gamma NO(-) macrophages were stimulated with IFN-gamma  + LPS alone or coincubated with 30 µM PD-98059, 30 µM SB-203580, or 0.075% DMSO for 18 h, and then the supernatant was subjected to nitrite assay. Results are means of 6 experiments. ***P < 0.001 vs. 2nd bar. NS, not significant (P > 0.05). B: Western blot with the iNOS antibody. Macrophages were stimulated with IFN-gamma  + LPS with and without PD-98059 (PB) or SB-203580 (SB) for 18 h, and the nuclear-free lysate was immunoblotted with iNOS antibody. Result is representative of 3 independent experiments.

p38mapk inhibitor (SB-203580) augments NO<SUB>2</SUB><SUP>−</SUP> independent of IFN-gamma + LPS-induced TNF-alpha expression. To determine the role of p38mapk on NO<SUB>2</SUB><SUP>−</SUP> and iNOS protein expression induced by IFN-gamma  + LPS, we pretreated RAW 264.7gamma NO(-) cells with SB-203580 (30 µM) for 1 h, costimulated the cells with IFN-gamma  + LPS in the presence of the inhibitor for 18 h, and then quantified NO<SUB>2</SUB><SUP>−</SUP> accumulation in culture supernatants and lysis of cells for immunoblotting for iNOS protein. In contrast to that observed with MEK1 inhibition, inhibition of p38mapk augmented NO<SUB>2</SUB><SUP>−</SUP> production, indicating that p38mapk plays an inhibitory role in NO· induction (Fig. 4A). There was a small increase in the amount of iNOS protein expression in the presence of SB-203580 (Fig. 4B).

Potential paracrine and autocrine role of TNF-alpha and IL-1beta in IFN-gamma + LPS induction of iNOS-NO·. LPS is known to induce the expression of a number of cytokines such as TNF-alpha , which also has the capacity, in conjunction with IFN-gamma , to induce iNOS expression. Thus a possible mechanism by which ERK may enhance IFN-gamma  + LPS-induced expression of NO· is by signaling the expression of cytokines such as TNF-alpha with IFN-gamma  + LPS stimulation. We therefore measured the amount of TNF-alpha protein levels in macrophages stimulated with IFN-gamma  + LPS in the presence of MEK1-ERK inhibition by PD-98059. As shown in Fig. 5A, stimulation of cells with IFN-gamma  + LPS resulted in a significant increase in the amount of TNF-alpha measured in the supernatant. Coincubation with PD-98059 substantially inhibited TNF-alpha protein expression in the supernatant of RAW 264.7gamma NO(-) cells. Interestingly, despite the fact that SB-203580 augmented NO<SUB>2</SUB><SUP>−</SUP> expression by IFN-gamma  + LPS, it also inhibited TNF-alpha protein expression, although the level of inhibition was not as marked as that seen with MEK1-ERK inhibition (Fig. 5A). The vehicle DMSO at a concentration equivalent to 30 µM PD-98059 (0.075%) had no effect on stimulated TNF-alpha expression (Fig. 5A). To determine whether induction of NO· by IFN-gamma  + LPS is due to an autocrine or paracrine effect of TNF-alpha and/or IL-1beta , we neutralized TNF-alpha , IL-1beta , or both with neutralizing antibodies in the presence of IFN-gamma  + LPS stimulation. RAW 264.7 cells were coincubated with IFN-gamma  + LPS along with neutralizing concentrations of anti-TNF-alpha antibody (2.5-5 µg/ml), anti-IL-1beta antibody (2.5-5 µg/ml), anti-TNF-alpha antibody + anti-IL-1beta antibody (5 µg/ml each), or nonimmune IgG isotype antibody (2.5-10 µg/ml). After 18 h of incubation, the supernatants were assayed for NO<SUB>2</SUB><SUP>−</SUP>. As shown in Fig. 5B, incubation with neutralizing anti-TNF-alpha antibody resulted in a partial inhibition of NO<SUB>2</SUB><SUP>−</SUP>, with ~33% inhibition at 5 µg/ml anti-TNF-alpha IgG. In contrast, neither anti-IL-1beta antibody nor nonimmune IgG isotype had any inhibitory effect. Neutralization of TNF-alpha and IL-1beta was not significantly different from neutralization of TNF-alpha alone.


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Fig. 5.   A: IFN-gamma  + LPS-induced tumor necrosis factor-alpha (TNF-alpha ) protein expression in the presence of PD-98059 or SB-203580. TNF-alpha protein levels were measured by ELISA of the supernatant of RAW 264.7gamma NO(-) cells after 18 h of stimulation in the presence or absence of the inhibitors. Results are means of 3 independent experiments. ***P < 0.001 vs. 2nd bar. B: effect of neutralizing TNF-alpha and/or IL-1beta on IFN-gamma  + LPS induction of NO·. RAW 264.7 cells were incubated with IFN-gamma  + LPS with or without anti-TNF-alpha antibody (2.5-5 µg/ml), anti-IL-1beta antibody (2.5-5 µg/ml), anti-TNF-alpha antibody + anti-IL-1beta antibody (5 µg/ml each), or nonimmune IgG isotype (2.5-10 µg/ml) for 18 h, and the culture supernatants were assayed for NO<SUB>2</SUB><SUP>−</SUP>. Results are means of 3 independent experiments. ***P < 0.001 vs. 2nd bar.

DN-MKK4, but not DN-MEKK1, inhibits iNOS promoter activation by IFN-gamma + LPS. A pharmacological inhibitor of the JNK pathway is not available commercially. To investigate the role of the MEKK1-MKK4-JNK pathway in iNOS expression by LPS, we used a reporter gene assay in which RAW 264.7gamma NO(-) cells were cotransfected with 0.3 µg of an iNOS promoter-luciferase plasmid (iNOS-luc) and DN-MEKK1 or DN-MKK4 (10, 28, 45, 58). After transfection and growth for 72 h, the cells were stimulated with IFN-gamma (10 U/ml) + LPS (l ng/ml) for 8 h, and luciferase activity in cell lysates was measured. As shown in Fig. 6A, transfection of the iNOS-luc alone followed by IFN-gamma  + LPS stimulation resulted in an ~20-fold induction of luciferase activity compared with unstimulated cells. However, in cells cotransfected with the DN-MKK4, there was a significant decrease in luciferase activity (Fig. 6A). We next determined the effects of DN-MEKK1 on iNOS promoter regulation by IFN-gamma  + LPS. After transfection of iNOS-luc with or without DN-MEKK1, RAW 264.7gamma NO(-) cells were stimulated with IFN-gamma (10 U/ml) + LPS (l ng/ml) for 8 h. In contrast to the inhibitory effects observed with the DN-MKK4 construct, DN-MEKK1 did not inhibit iNOS promoter activity after stimulation with IFN-gamma  + LPS (Fig. 6B).


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Fig. 6.   iNOS promoter-luciferase reporter assays of macrophages cotransfected with dominant-negative (DN)-MKK4 or DN-MEKK1. A: RAW 264.7gamma NO(-) macrophages were transfected with 0.3 µg of iNOS-luc plasmid and 2 µg of DN-MKK4 or 2 µg LNCX vector with the Lipofectamine reagent and then stimulated with IFN-gamma  + LPS. Nuclear-free lysates were then measured for luciferase activity. Results are reported as magnitude increase in luciferase activity and are normalized for beta -galactosidase activity. Results are means of 3 experiments. *P < 0.05 vs. 2nd bar. B: macrophages were transfected with 0.3 µg of iNOS-luc plasmid and 2 µg of DN-MEKK1 or 2 µg pcDNA3 vector and then stimulated with IFN-gamma  + LPS for 8 h, and luciferase activity was measured. Results are means of 3 experiments. NS, P > 0.05 vs. 2nd bar.

DN-c-Jun does not inhibit iNOS promoter activity by IFN-gamma + LPS. Preliminary work by others with truncated forms of the iNOS-luc constructs suggested that AP-1 was not involved in its regulation by IFN-gamma  + LPS (30, 55). However, the significant inhibition of iNOS promoter activity by DN-MKK4 in IFN-gamma  + LPS-treated cells raises the question of how the JNK pathway may be regulating iNOS expression by LPS. One possibility is that c-Jun participates as a trans-activating factor, via AP-1 or in association with another transcription factor. Thus we tested the effects of a DBD-c-Jun, which contains the DNA-binding domain but lacks the transactivating domain, in the iNOS-luc reporter assay. With IFN-gamma  + LPS stimulation, there was no significant difference in iNOS promoter activity in the presence or absence of DBD-c-Jun (Fig. 7). Therefore, although the MKK4-JNK pathway plays a positive regulatory role in the promoter activity of iNOS stimulated with IFN-gamma  + LPS, c-Jun does not appear to be involved.


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Fig. 7.   iNOS luciferase reporter assay of macrophages cotransfected with dominant-negative c-Jun (DBD-c-Jun). RAW 264.7gamma NO(-) cells were transfected with 0.3 µg of iNOS-luc plasmid and 2 µg of DBD-c-Jun or 2 µg LNCX vector with the Lipofectamine reagent and then stimulated with IFN-gamma  + LPS. Nuclear-free lysates were then measured for luciferase activity and normalized for beta -galactosidase activity. Results are means of 3 experiments. NS, P > 0.05 vs. 2nd bar.

SB-203580 augments JNK phosphorylation and activation by IFN-gamma + LPS. p38mapk has been shown to inhibit ERK activation (23). Thus the dichotomy we observed in IFN-gamma  + LPS-stimulated iNOS induction showing the inhibitory effect of p38mapk vs. the enhancing effect of JNK and ERK raises the possibility that MAPK interaction may also be occurring. Therefore, we examined the effects of SB-203580 on IFN-gamma  + LPS-induced JNK and ERK phosphorylation and the effects of PD-98059 on the phosphorylation of JNK and p38mapk. RAW 264.7gamma NO(-) cells were pretreated with SB-203580 (15 and 30 µM) or PD-98059 (15 and 30 µM) for 1 h and then costimulated with LPS (100 ng/ml) + IFN-gamma (10 U/ml) for an additional 1 h. After the cells were lysed and the lysates were normalized for protein content, a portion was separated by SDS-PAGE and immunoblotted with phospho-specific JNK, ERK, or p38mapk antibody. As expected, PD-98059 inhibited phosphorylation of ERK (Fig. 8A). PD-98059 also did not affect TXY phosphorylation of p38mapk or JNK by LPS + IFN-gamma (Fig. 8, B and C). As shown in Fig. 8B, SB-203580 is not expected to inhibit the phosphorylation of p38mapk in a dose-responsive fashion, although we have shown that it does inhibit p38mapk activity (5). Inhibition of p38mapk with SB-203580 also did not affect the phosphorylation of ERK by LPS + IFN-gamma (Fig. 8A). However, SB-203580 substantially and consistently increased the phosphorylation of Thr-183 and Tyr-185 residues of both JNK isoforms (Fig. 8C). To determine whether this augmentation in phosphorylation of JNK by SB-203580 is also accompanied by an increase in JNK activity, another portion of the whole cell lysate was subjected to a solid-phase in vitro kinase assay with c-Jun-GST beads as the substrate. As shown in Fig. 9 (top), IFN-gamma  + LPS-induced JNK activity was significantly augmented by SB-203580. Because JNK activation also correlates with the binding of JNK to c-Jun (4), we immunoblotted the nitrocellulose membrane with p46 JNK1 antibody. As shown in Fig. 9, bottom, the amount of p46 JNK1 bound to c-Jun correlated with JNK activity, although the amount was modest.


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Fig. 8.   Enhancement of IFN-gamma  + LPS-induced JNK phosphorylation by SB-203580. RAW 264.7gamma NO(-) macrophages were treated with SB-203580 (15 and 30 µM), PD-98059 (15 and 30 µM), or the vehicle DMSO in an amount equivalent to 30 µM PD-98059 (0.075%) for 1 h and then costimulated with IFN-gamma (10 U/ml) + LPS (100 ng/ml) for 1 h, and nuclear-free whole cell lysates were subjected to Western blotting with phospho-specific ERK (A), p38mapk (B), and JNK (C) antibodies. Result is representative of 2 independent experiments.



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Fig. 9.   Augmentation of IFN-gamma  + LPS-induced JNK activity by SB-203580. RAW 264.7gamma NO(-) macrophages were treated with SB-203580 (15 and 30 µM) or equivalent amount of the vehicle DMSO (0.03 and 0.06%) for 1 h and costimulated with IFN-gamma (10 U/ml) + LPS (100 ng/ml) for 1 h, and then a solid-phase in vitro kinase assay with c-Jun-GST-Sepharose beads was carried out in the presence of [gamma -32P]ATP (top). Bottom: immunoblot of the nitrocellulose membrane at top with p46 JNK1 antibody. wcl, Whole cell lysate. Result is representative of 2 independent experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

A greater understanding of the signaling pathways involved in the regulation of iNOS by LPS may have potential therapeutic implications in a number of disorders in which LPS and NO· play important deleterious or beneficial roles, such as septic shock and host defense against microbes. Although others have also investigated the role of the MAPKs in iNOS regulation by various stimuli, the role of the JNK pathway has not been systematically examined with LPS stimulation. A broad conclusion that can be drawn from these works is that, depending on the stimulus and cell type, MAPKs may play a positive, negative, or neutral regulatory role in iNOS expression (2, 7, 14, 18, 47, 51). Bhat and coworkers (2) examined the role of ERK and p38mapk in the expression of NO· by LPS in primary glial cells. Similar to our findings, they showed that inhibition of MEK1-ERK suppressed NO· and iNOS expression. However, in contrast to the present study, they found that p38mapk inhibition also inhibited NO· expression by LPS. Chen and Wang (6) recently reported that p38mapk augmented iNOS induction by LPS but that ERK was not involved. Although this finding is opposite to what we observed, they used an RAW 264.7 cell lineage distinct from that used in this study. In addition, these investigators stimulated their macrophages with an amount of LPS that was 1 × 103-1 × 106 greater than the amount we used. Further evidence that the experimental conditions were different is that they noted maximal activation of the MAPKs after 10 min of stimulation; in contrast, we did not see any MAPK activation until after ~30 min of stimulation, which peaked only after 60 min of stimulation. Ajizian et al. (1) noted that inhibition of p38mapk or ERK blocked iNOS and TNF-alpha accumulation in macrophages stimulated with IFN-gamma  + LPS. Although their data are consistent with ours in regard to TNF-alpha production, their data are opposite to our observation of iNOS-NO· augmentation with inhibition of p38mapk. Although this discrepancy may also be due to the fact that different lineages of RAW 264.7 cells were used (39), these investigators measured only iNOS mRNA, and p38mapk has been shown to also have posttranscriptional effects (27). Others have also noted that the role of p38mapk is cell and/or stimulus specific. For example, Da Silva and coworkers (7) demonstrated that p38mapk is necessary, but not sufficient, for iNOS induction by TNF-alpha  + IL-1alpha stimulation in mouse astrocytes. In contrast, Guan et al. (18) found that p38mapk inhibited NO· expression by IL-1beta in rodent mesangial cells, an effect similar to our observation with IFN-gamma  + LPS stimulation.

Here we have demonstrated that the MEK1-ERK and MKK4-JNK pathways augmented and p38mapk inhibited IFN-gamma  + LPS induction of iNOS/NO·. These modulatory effects of the MAPKs raise the question of how they ultimately affect the transcription of iNOS with IFN-gamma  + LPS stimulation. We previously showed that ERK mediated TNF-alpha expression on engagement of the Fcgamma receptor (44). Thus we reasoned that a possible mechanism by which ERK augments iNOS induction is through its ability to signal LPS induction of TNF-alpha or IL-1beta , cytokines that are capable of inducing NO· expression. Our demonstration that TNF-alpha expression with IFN-gamma  + LPS stimulation is ERK dependent would lend credence to this hypothesis. The fact that there was only modest inhibition of NO<SUB>2</SUB><SUP>−</SUP> expression by PD-90859 is perhaps due to the low copy number of p55 TNF receptor on the surface of RAW 264.7 cells (12). This finding was corroborated by experiments in which neutralization of TNF-alpha with anti-TNF-alpha antibody resulted in a reduction of IFN-gamma  + LPS-induced NO<SUB>2</SUB><SUP>−</SUP> expression by ~33% (Fig. 5B), a level of inhibition similar to that seen with PD-98059 (Fig. 4A). Neutralization of IL-1beta with anti-IL-1beta antibody had no effect on IFN-gamma  + LPS-induced NO<SUB>2</SUB><SUP>−</SUP> expression, consistent with the fact that RAW 264.7 cells respond very poorly to IL-1beta (25).

Previous works have shown that the three nonconsensus AP-1 sites in the 5'-flanking region of the iNOS promoter were not involved in enhancing iNOS transcription by LPS (30, 56). The lack of inhibition of iNOS promoter activity by DBD-c-Jun would substantiate this finding. Because previous studies have shown that NF-kappa B is a critical regulator of iNOS transcription by LPS, potential mechanisms for the role of MKK4-JNK and their feasibility are as follows: 1) JNK itself has been shown to bind the c-Rel component of NF-kappa B and to enhance NF-kappa B activation (37). Moreover, in an overexpression system, transfected JNK was able to activate a CAT reporter gene driven by two kappa B elements (37). In a recent report, Janssen-Heininger and coworkers (22) showed that the JNK pathway was important in the activation of NF-kappa B by oxidants and TNF-alpha in lung epithelial cells. 2) Another possibility, although not mutually exclusive, is that the bZIP region of c-Jun may bind directly to the p65 subunit of NF-kappa B to enhance the binding of NF-kappa B to its kappa B element (48). Although DBD-c-Jun would be expected to inhibit AP-1 activity, it is not known whether it would also be capable of inhibiting any NF-kappa B-c-Jun interaction, because the mutated c-Jun still contains the bZIP region that is capable of binding the p65 subunit (48). 3) Although JNK is best known for transactivating c-Jun, it may also directly or indirectly activate a number of other transcription factors, including ATF-2, Elk-1, cAMP-responsive element binding protein, and NF-kappa B (19, 22, 37, 52). Thus JNK may activate other uncharacterized transcription factors that may be directly or indirectly involved in transcriptional control of LPS-induced iNOS. For example, Welsh (51) showed that JNK1 enhanced IL-1beta induction of iNOS by activating ATF-2, which then physically cooperated with NF-kappa B to facilitate binding of NF-kappa B to its kappa B sites on the iNOS promoter. More recently, Sawada and colleagues (46) suggested that, with IL-6 stimulation of iNOS expression, an octomer-binding protein may physically associate with and synergize NF-kappa B activity.

We previously found that, in NIH/3T3 fibroblasts, DN-MEKK1 inhibited iNOS promoter activity with IFN-gamma  + TNF-alpha stimulation (5). However, we did not find such an effect in RAW 264.7gamma NO(-) cells stimulated with IFN-gamma  + LPS. Although MEKK1 has been implicated in the activation of JNK, more recent studies have shown that other MAPKKK such as germinal center kinases, mixed-lineage kinases, p21-activated kinases, tyrosine phenol-lyases, SPRK, and other isoforms of MEKK may also participate in the activation of the JNK pathway, depending on the cell types, stimuli, and culture conditions (13, 42).

We found an inhibitory role for p38mapk in the induction of iNOS-NO· by IFN-gamma  + LPS. This inhibition by p38mapk is clearly not due to an inhibition of IFN gamma  + LPS induction of TNF-alpha , because p38mapk also enhanced IFN-gamma  + LPS-induced TNF-alpha protein expression. Because the MKK4-JNK and MEK1-ERK pathways augmented iNOS induction by IFN-gamma  + LPS, we reasoned that a plausible mechanism of how p38mapk inhibited IFN-gamma  + LPS-induced NO<SUB>2</SUB><SUP>−</SUP> expression is by inhibiting JNK and/or ERK activation. There is precedence for this notion that one MAPK may affect the activity of another, because p38mapk has been shown to inhibit ERK activation by platelet-derived growth factor or tryptase in airway smooth muscle cells (23). We therefore explored this possibility and demonstrated that inhibition of p38mapk substantially augmented IFN-gamma  + LPS-induced JNK phosphorylation and activation, providing further insight into the realm of MAPK interactions. Recently, p38mapk was found to inhibit ERK1- and ERK2-mediated growth of Kaposi's sarcoma cells (38). Marinissen and colleagues (32) showed an even more complex interaction between JNK, p38mapk, and ERK5 in the activation of the c-jun promoter after engagement of muscarinic M1 receptors.

In summary, we have shown that ERK and JNK signal transduction pathways augmented and p38mapk inhibited iNOS-induction by IFN-gamma  + LPS. Cotransfection studies with the iNOS-luc reporter gene and dominant-inhibitory mutant proteins of the MEKK1-MKK4-JNK-c-Jun pathway suggest that although MKK4-JNK appears to augment IFN-gamma  + LPS-induced iNOS promoter activity, MEKK1 and c-Jun do not. These findings imply that, in RAW 264.7gamma NO(-) cells, another MAPKKK activates MKK4 with IFN-gamma  + LPS stimulation and that the role of MKK4-JNK is due to an AP-1-independent event. Future studies in the manipulation of these signaling pathways in which LPS and NO· play critical roles may shed new light on various disease processes such as endotoxin-mediated septic shock.


    ACKNOWLEDGEMENTS

We thank Kristin R. Morris, Linda K. Remigio, and Cheryl Leu for expert technical assistance. We are also grateful to Boyd Jacobson, Barry Silverstein, and Nadia de Steckelberg for illustrative assistance.


    FOOTNOTES

E. D. Chan is supported by National Heart, Lung, and Blood Institute Clinical Investigator Development Award 1K08 HL-036250-01, the Bettina Garthwaite Lowerre Foundation for Mycobacteriology Research, and the Parke-Davis Atorvastatin Research Grant. D. W. H. Riches is supported by National Heart, Lung, and Blood Institute Grant HL-55549 and Specialized Center of Research Grant HL-56556.

Address for reprint requests and other correspondence: E. D. Chan, K613e, Goodman Bldg., National Jewish Medical and Research Center, 1400 Jackson St., Denver, CO 80206 (E-mail: chane{at}njc.org).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 11 July 2000; accepted in final form 25 September 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1.   Ajizian, SJ, English BK, and Meals EA. Specific inhibitors of p38 and extracellular signal-regulated kinase-mitogen-activated protein kinase pathways block inducible nitric oxide synthase and tumor necrosis factor accumulation in murine macrophages stimulated with lipopolysaccharide and interferon-gamma . J Infect Dis 179: 939-944, 1999[ISI][Medline].

2.   Bhat, NR, Zhang P, Lee JC, and Hogan EL. Extracellular signal-regulated kinase and p38 subgroups of mitogen-activated protein kinases regulate inducible nitric oxide synthase and tumor necrosis factor-alpha gene expression in endotoxin-stimulated primary glial cultures. J Neurosci 18: 1633-1641, 1998[Abstract/Free Full Text].

3.   Chan, ED, and Riches DWH Potential role of the JNK/SAPK signal transduction pathway in the induction of iNOS by TNF-alpha . Biochem Biophys Res Commun 253: 790-796, 1998[ISI][Medline].

4.   Chan, ED, Winston BW, Jarpe MB, Wynes MW, and Riches DWH Preferential activation of the p46 isoform of JNK/SAPK in mouse macrophages by TNF-alpha . Proc Natl Acad Sci USA 94: 13169-13174, 1997[Abstract/Free Full Text].

5.   Chan, ED, Winston BW, Uh S-T, Wynes MW, Rose DM, and Riches DWH Evaluation of the role of mitogen-activated protein kinases in the expression of inducible nitric oxide synthase by IFN-gamma and TNF-alpha in mouse macrophages. J Immunol 162: 415-422, 1999[Abstract/Free Full Text].

6.   Chen, C-C, and Wang J-K. p38 but not p44/42 mitogen-activated protein kinase is required for nitric oxide synthase induction mediated by lipopolysaccharide in RAW 264.7 macrophages. Mol Pharmacol 55: 481-488, 1999[Abstract/Free Full Text].

7.   Da Silva, J, Pierrat B, Mary JL, and Lesslauer W. Blockade of p38 mitogen-activated protein kinase pathway inhibits inducible nitric-oxide synthase expression in mouse astrocytes. J Biol Chem 272: 28373-28380, 1997[Abstract/Free Full Text].

8.   Deacon, K, and Blank JL. MEK kinase 3 directly activates MKK6 and MKK7, specific activators of the p38 and c-Jun NH2-terminal kinases. J Biol Chem 274: 16604-16610, 1999[Abstract/Free Full Text].

9.   Dérijard, B, Hibi M, Wu I-H, Barrett T, Su B, Deng T, Karin M, and Davis RJ. JNK1: a protein kinase stimulated by UV light and Ha-Ras that binds and phosphorylates the c-Jun activation domain. Cell 76: 1025-1037, 1994[ISI][Medline].

10.   Derijard, B, Raingeaud J, Barrett T, Wu I-H, Han J, Ulevitch RJ, and Davis RJ. Independent human MAP kinase signal transduction pathways defined by MEK and MKK isoforms. Science 267: 682-685, 1995[ISI][Medline].

11.   Ding, AH, Nathan CF, and Stuehr DJ. Release of reactive nitrogen intermediates and reactive oxygen intermediates from mouse peritoneal macrophages: comparison of activating cytokines and evidence for independent production. J Immunol 141: 2407-2412, 1988[Abstract/Free Full Text].

12.   Ding, AH, Sanchez E, Srimal S, and Nathan CF. Macrophages rapidly internalize their receptors in response to bacterial lipopolysaccharide. J Biol Chem 264: 3924-3929, 1989[Abstract/Free Full Text].

13.   Fanger, GR, Gerwins P, Widmann C, Jarpe MB, and Johnson GL. MEKKs, GCKs, MLKs, PAKs, TAKs, and Tpls: upstream regulators of the c-Jun amino-terminal kinases? Curr Opin Genet Dev 7: 67-74, 1997[ISI][Medline].

14.   Finder, JD, Litz JL, Blaskovich MA, McGuire TF, Qian Y, Hamilton AD, Davies P, and Sebti SM. Inhibition of protein geranylgeranylation causes a superinduction of nitric-oxide synthase-2 by interleukin-1beta in vascular smooth muscle cells. J Biol Chem 272: 13484-13488, 1997[Abstract/Free Full Text].

15.   Gao, J, Morrison DC, Parmely TJ, Russell SW, and Murphy WJ. An interferon-gamma -activated site (GAS) is necessary for full expression of the mouse iNOS gene in response to interferon-gamma and lipopolysaccharide. J Biol Chem 272: 1226-1230, 1997[Abstract/Free Full Text].

16.   Goldring, CEP, Narayanan R, Lagadec P, and Jeannin J-F. Transcriptional inhibition of the inducible nitric oxide synthase gene by competitive binding of NF-kappa B/REL proteins. Biochem Biophys Res Commun 209: 73-79, 1995[ISI][Medline].

17.   Goldring, CEP, Reveneau S, Algarte M, and Jeannin J-F. In vivo footprinting of the mouse inducible nitric oxide synthase gene: inducible protein occupation of numerous sites including Oct and NF-IL-6. Nucleic Acids Res 24: 1682-1687, 1996[Abstract/Free Full Text].

18.   Guan, Z, Baier LD, and Morrison AR. p38 mitogen-activated protein kinase downregulates nitric oxide and upregulates prostaglandin E2 biosynthesis stimulated by interleukin-1beta . J Biol Chem 272: 8083-8089, 1997[Abstract/Free Full Text].

19.   Gupta, S, Campbell D, Derijard B, and Davis R. Transcription factor ATF2 regulation by the JNK signal transduction pathway. Science 267: 389-393, 1995[ISI][Medline].

20.   Han, J, Lee JD, Bibbs L, and Ulevitch RJ. A MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells. Science 265: 808-811, 1994[ISI][Medline].

21.   Hibi, M, Lin A, Smeal T, Minden A, and Karin M. Identification of an oncoprotein- and UV-responsive protein kinase that binds and potentiates the c-Jun activation domain. Genes Dev 7: 2135-2148, 1993[Abstract].

22.   Janssen-Heininger, YMW, Macara I, and Mossman BT. Cooperativity between oxidants and tumor necrosis factor in the activation of nuclear factor (NF)-kappa B: requirement of Ras/mitogen-activated protein kinases in the activation of NF-kappa B by oxidants. Am J Respir Cell Mol Biol 20: 942-952, 1999[Abstract/Free Full Text].

23.   Jones, CA, and Brown JK. p38 inhibition increases ERK1/ERK2 kinase activity and DNA synthesis in airway smooth muscle cells: evidence for cross-talk between mitogen-activated protein (MAP) kinase cascades (Abstract). Am J Respir Crit Care Med 159: A722, 1999[ISI].

24.   Kamijo, R, Harada H, Matsuyama T, Bosland M, Gerecitano J, Shapiro D, Le J, Koh SI, Kimura T, Green S, Mak TW, Taniguchi T, and Vilcek J. Requirement for transcription factor IRF-1 in NO synthase induction in macrophages. Science 263: 1612-1615, 1994[ISI][Medline].

25.   Kim, YM, and Son K. A nitric oxide production bioassay for interferon-gamma . J Immunol Methods 198: 203-209, 1996[ISI][Medline].

26.   Kyriakis, JM, Banerjee P, Nikolakaki E, Dai T, Rubie EA, Ahmad MF, Avruch J, and Woodgett JR. The stress-activated protein kinase subfamily of c-Jun kinases. Nature 369: 156-160, 1994[ISI][Medline].

27.   Lee, JC, Layton JT, McDonnel PC, Gallagher TF, Kumar S, Green D, McNulty D, Blumenthal MJ, Heys JR, and Landvatter SW. A protein kinase involved in the regulation of inflammatory cytokine biosynthesis. Nature 372: 739-746, 1994[ISI][Medline].

28.   Lin, A, Minden A, Martinetto H, Claret F-X, Lange-Carter C, Mercurio F, Johnson GL, and Karin M. Identification of a dual specificity kinase that activates the Jun kinases and p38-Mpk2. Science 268: 286-290, 1995[ISI][Medline].

29.   Lorsbach, RB, Murphy WJ, Lowenstein CJ, Snyder SH, and Russell SW. Expression of the nitric oxide synthase gene in mouse macrophages activated for tumor cell killing. J Biol Chem 268: 1908-1913, 1993[Abstract/Free Full Text].

30.   Lowenstein, CJ, Alley EW, Raval P, Snowman AD, Snyder SH, Russell SW, and Murphy WJ. Macrophage nitric oxide synthase gene: two upstream regions mediate induction by interferon-gamma and lipopolysaccharide. Proc Natl Acad Sci USA 90: 9730-9734, 1993[Abstract].

31.   Lyons, CR, Orloff GJ, and Cunningham JM. Molecular cloning and functional expression of an inducible nitric oxide synthase from a murine macrophage cell line. J Biol Chem 267: 6370-6374, 1992[Abstract/Free Full Text].

32.   Marinissen, MJ, Chiariello M, Pallante M, and Gutkind JS. A network of mitogen-activated protein kinases links G protein-coupled receptors to the c-jun promoter: a role for c-Jun NH2-terminal kinase, p38s, and extracellular signal-regulated kinase 5. Mol Cell Biol 19: 4289-4301, 1999[Abstract/Free Full Text].

33.   Marletta, MA. Nitric oxide synthase: aspects concerning structure and catalysis. Cell 78: 927-930, 1994[ISI][Medline].

34.   Martin, E, Nathan C, and Xie QW. Role of interferon regulatory factor 1 in induction of nitric oxide synthase. J Exp Med 180: 977-984, 1994[Abstract].

35.   Melillo, G, Cox GW, Biragyn A, Sheffler LA, and Varesio L. Regulation of nitric-oxide synthase mRNA expression by interferon-gamma and picolinic acid. J Biol Chem 269: 8128-8133, 1994[Abstract/Free Full Text].

36.   Melillo, G, Musso T, Sica A, Taylor LS, Cox GW, and Varesio L. A hypoxia-responsive element mediates a novel pathway of activation of the inducible nitric oxide synthase promoter. J Exp Med 182: 1683-1693, 1995[Abstract].

37.   Meyer, CF, Wang X, Chang C, Templeton D, and Tan TH. Interaction between c-Rel and the mitogen-activated protein kinase kinase kinase 1 signaling cascade in mediating kappa B enhancer activation. J Biol Chem 271: 8971-8976, 1996[Abstract/Free Full Text].

38.   Murakami-Mori, K, Mori S, and Shuji N. p38MAP kinase is a negative regulator for ERK1/2-mediated growth of AIDS-associated Kaposi's sarcoma cells. Biochem Biophys Res Commun 264: 676-682, 1999[ISI][Medline].

39.   Murphy, WJ, Muroi M, Zhang X, Suzuki T, and Russell SW. Both a basal and an enhancer IB element is required for full induction of the mouse inducible nitric oxide synthase gene. J Endotoxin Res 3: 381-393, 1996[ISI].

40.   Nathan, C, and Xie QW. Regulation of biosynthesis of nitric oxide. J Biol Chem 269: 13725-13728, 1994[Free Full Text].

41.   Nussler, AK, and Billiar TR. Inflammation, immunoregulation, and inducible nitric oxide synthase. J Leukoc Biol 54: 171-178, 1993[Abstract].

42.   Rana, A, Gallo K, Godowski P, Hirai S, Ohno S, Zon L, Kyriakis JM, and Avruch J. The mixed lineage kinase SPRK phosphorylates and activates the stress-activated protein kinase activator, SEK-1. J Biol Chem 271: 19025-19028, 1996[Abstract/Free Full Text].

43.   Riches, DWH, Chan ED, Zahradka EA, Winston BW, Remigio LK, and Lake FR. Cooperative signaling by tumor necrosis factor receptors CD120a (p55) and CD120b (p75) in the expression of nitric oxide and inducible nitric oxide synthase by mouse macrophages. J Biol Chem 273: 22800-22806, 1998[Abstract/Free Full Text].

44.   Rose, DM, Winston BW, Chan ED, Riches DWH, Gerwins P, Johnson GL, and Henson PM. Fcgamma receptor cross-linking activates p42, p38, and JNK/SAPK mitogen-activated protein kinases in murine macrophages. J Immunol 158: 3433-3438, 1997[Abstract].

45.   Sanchez, I, Hughes RT, Mayer BJ, Yee K, Woodgett JR, Avruch J, Kyriakis JM, and Zon LI. Role of SAPK/ERK kinase-1 in the stress-activated pathway regulating transcription factor c-Jun. Nature 372: 794-798, 1994[ISI][Medline].

46.   Sawada, T, Falk LA, Rao P, Murphy WJ, and Pluznik DH. IL-6 induction of protein-DNA complexes via a novel regulatory region of the inducible nitric oxide synthase gene promoter: role of octomer binding proteins. J Immunol 158: 5267-5276, 1997[Abstract].

47.   Singh, K, Balligand J-L, Fischer TA, Smith TW, and Kelly RA. Regulation of cytokine-inducible nitric oxide synthase in cardiac myocytes and microvascular endothelial cells: role of extracellular signal-regulated kinases 1 and 2 (ERK1/ERK2) and Stat1alpha . J Biol Chem 271: 1111-1117, 1996[Abstract/Free Full Text].

48.   Stein, B, Baldwin AS, Ballard DW, Greene WC, Angel P, and Herrlich P. Cross-coupling of the NF-kappa B p65 and Fos/Jun transcription factors produces potentiated biological function. EMBO J 12: 3879-3891, 1993[Abstract].

49.   Taylor, BS, de Vera ME, Ganster RW, Wang Q, Shapiro RA, Morris SMJ, Billiar TR, and Geller DA. Multiple NF-kappa B enhancer elements regulate cytokine induction of the human inducible nitric oxide synthase gene. J Biol Chem 273: 15148-15156, 1998[Abstract/Free Full Text].

50.   Towbin, H, Staehelin T, and Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76: 4350-4354, 1979[Abstract].

51.   Welsh, N. Interleukin-1beta -induced ceramide and diacylglycerol generation may lead to activation of the c-Jun NH2-terminal kinase and the transcription factor ATF2 in the insulin-producing cell line RINm5F. J Biol Chem 271: 8307-8312, 1996[Abstract/Free Full Text].

52.   Whitmarsh, AJ, Shore P, Sharrocks AD, and Davis RJ. Integration of MAP kinase signal transduction pathways at the serum response element. Science 269: 403-407, 1995[ISI][Medline].

53.   Winston, BW, Chan ED, Johnson GL, and Riches DWH Activation of p38mapk, MKK3, and MKK4 by tumor necrosis factor-alpha in mouse bone marrow-derived macrophages. J Immunol 159: 4491-4497, 1997[Abstract].

54.   Winston, BW, and Riches DWH Activation of p42mapk/erk2 following engagement of TNF receptor CD120a (p55) in mouse macrophages. J Immunol 155: 1525-1533, 1995[Abstract].

55.   Xie, Q, Whisnant R, and Nathan C. Promoter of the mouse gene encoding calcium-independent nitric oxide synthase confers inducibility by interferon-gamma and bacterial lipopolysaccharide. J Exp Med 177: 1779-1784, 1993[Abstract].

56.   Xie, Q-W, Cho HJ, Calaycay J, Mumford RA, Swiderek KM, Lee TD, Ding A, Troso T, and Nathan C. Cloning and characterization of inducible nitric oxide synthase from mouse macrophages. Science 256: 225-228, 1992[ISI][Medline].

57.   Xie, Q-W, Kashiwabara Y, and Nathan C. Role of transcription factor NF-kappa B/Rel in induction of nitric oxide synthase. J Biol Chem 269: 4705-4708, 1994[Abstract/Free Full Text].

58.   Yan, M, Dai T, Deak JC, Kyriakis JM, Zon LI, Woodgett JR, and Templeton DJ. Activation of stress-activated protein kinase by MEKK1 phosphorylation of its activator SEK1. Nature 372: 798-800, 1994[ISI][Medline].


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