Nuclear Factor- (cid:1) B and Mitogen-activated Protein Kinases Mediate Nitric Oxide-enhanced Transcriptional Expression of Interferon- (cid:2) *

Mitogen-activated protein (MAP) kinase and nuclear factor- (cid:1) B (NF- (cid:1) B) activation are critical for initiating the transcriptional expression of cytokines, cell adhesion molecules, and other factors in the macrophage immune response. Nitric oxide (NO), an endogenous free radical, is a product of macrophages that mediates inflammatory and cytotoxic processes in the immune system. Here we report the effects of NO on MAP kinase signaling and NF- (cid:1) B activation in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages and correlate these effects to the induction target genes, including interferon- (cid:2) (IFN- (cid:2) ) and I (cid:1) B- (cid:3) . LPS alone induced a rapid phosphorylation of the stress-activated MAP kinases: c-Jun N-terminal kinase (JNK) and p38. Simultaneous treatment with LPS and the NO donor, diethyl-amine NONOate (DEA/NO), enhanced and prolonged JNK and p38 phosphorylation. Similarly, DEA/NO prolonged the LPS-induced degradation of the NF- (cid:1) B inhibitory subunit, I (cid:1) B- (cid:3) , despite an increase in I (cid:1) B- (cid:3) mRNA levels. Whereas DEA/NO alone was sufficient to induce JNK and p38 phosphorylation, it was not oxyl-3-oxide; DTT, dithiothreitol; JNK, c-Jun N-terminal kinase; EMSA, electrophoretic mobility shift assay. Cayman Chemical) were dissolved in slightly basic (pH 10.0), ice-cold sterile water immediately prior to addition to cell culture. The compound 2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tet- ramethyl-1 H -imidazolyl-1-oxyl-3-oxide (carboxy-PTIO; Cayman Chem-ical) was dissolved in water immediately prior to use. Both DEA/NO and MAHMA/NO were examined for their effect on macrophage viabil-ity using the trypan blue dye exclusion assay, and each donor was determined to be negligible under the conditions used in the experiments. Protein Extraction— Following treatment with experimental agents, cells were washed with cold phosphate-buffered saline, scraped, and collected by centrifugation. For total proteins, cell pellets were resuspended in lysis buffer (10 m M Tris, pH 7.05, 50 m M NaCl, 30 m M sodium pyrophosphate, 2 m M EDTA, 50 m M NaF, 1% (v/v) Triton X-100; 1 m M phenylmethylsulfonyl fluoride, 1 (cid:4) g/ml aprotonin, 1 (cid:4) g/ml leupeptin, 1 (cid:4) g/ml pepstatin A). Samples were incubated on ice for 15 min then centrifuged at 14,000 (cid:3) g for 15 min at 4 °C to remove debris, and supernatant was stored at (cid:4) 80 °C. Protein concentrations were determined by the Bio-Rad protein assay, and samples were diluted to 2 (cid:4) g/ (cid:4) l. Nuclear extracts were prepared as previously described, with slight modifications. Briefly, fresh cell pellets were resuspended in 800 (cid:4) l of ice-cold buffer A (10 m M M M end-labeled using [ (cid:5) - 32 P]ATP, 7,000 Ci/mmol (ICN), and T4 polynucleotide kinase (Promega). Assays were performed according to an established method, with slight variations (27). Briefly, 5 (cid:4) g of nuclear protein extract incubated for 30 min room in binding 0.025% Igepal CA-630, 0.05 m M EDTA, 0.05 m M EGTA, m M DTT, 0.5 m M phenylmethylsulfonyl containing 10 (cid:4) g of bovine serum albumin and 1 (cid:4) g of double-stranded poly(dI (cid:1) dC). 100,000 cpm of radiolabeled consensus oligonucleotide ( (cid:5) 0.1 pmol) was subsequently added to each sample, and incubated at room temperature for an additional 30 min. Samples labeled cold com- petitor indicate the addition of a 100-fold excess of unlabeled (cid:3) B oligonucleotide. Protein-DNA complexes were subsequently resolved in a 5% native Tris/taurine-buffered gel. Gels were dried and exposed to autoradiographic film at (cid:4) 80 °C. Ribonuclease Protection Assay— Following treatment with experimental agents, cells were washed with cold phosphate-buffered saline, harvested using a cell scraper and collected by centrifugation. Total RNA was extracted using the RNeasy Mini kit (Qiagen). RNA was quantified by absorbance

Inducible expression of a variety of genes is mediated through mitogen-activated protein (MAP) 1 kinase signaling pathways. MAP kinases regulate the phosphorylation and activation of transcription factors, including c-Jun, activating transcription factor-2, and Elk-1. The activation of MAP kinase pathways is critical in the induced expression of inflammatory cytokines. For example, MAP kinases regulate the transcrip-tional expression of the genes encoding tumor necrosis factor-␣, and interferon-␤ (IFN-␤) (1)(2)(3). In addition, MAP kinase pathways have been demonstrated to contribute to the activation of the ubiquitous transcription factor, nuclear factor-B (NF-B) (4,5). The NF-B pathway also has a critical role in the immune system, regulating both innate and adaptive responses. NF-B is essential for the inducible expression of cytokines, receptors for immune recognition, cell adhesion proteins, and the genes encoding inducible nitric-oxide synthase (iNOS) and cyclooxygenase-2 (6).
Although NF-B is comprised of a family of homologous DNA-binding proteins, it is prototypically represented as a heterodimer of p50 and p65 (RelA) subunits. Each subunit contains a conserved Rel homology domain, which encompasses the dimerization and DNA-binding motifs, and a nuclear localization signal (7,8). Transcriptional enhancement is conferred by the transactivation domain of NF-B, which is encoded at the C-terminal end of the p65 subunit (9). In nonstimulated cells, NF-B is retained in the cytoplasm in an inactive complex through interactions with the inhibitory protein IB (10,11). NF-B can be activated by a number of immunological stimuli, including the bacterial product, lipopolysaccharide (LPS), cytokines, and double-stranded RNA. Treatment with an appropriate stimulus induces rapid phosphorylation of IB on two specific serine residues (Ser-32 and Ser-36 for IB-␣) (12)(13)(14). Subsequent ubiquitination of IB and degradation by the 26 S proteasome releases the inhibition on NF-B, which then translocates to the nucleus and activates transcription of target genes (15)(16)(17)(18).
Transcriptional expression of IFN-␤ is regulated by an enhancer region located 55-105 bp upstream of the transcription start site (ϩ1), and which is highly conserved between human and mouse (19). Induction of IFN-␤ mRNA does not require de novo protein synthesis, but depends on existing transcription factors, which assemble into an enhanceosome complex at specific positive regulatory domains within the IFN-␤ promoter. The activation of NF-B and MAP kinase signaling pathways are critical for the formation of the IFN-␤ enhanceosome, because NF-B and a heterodimer of c-Jun/activating transcription factor-2 bind to positive regulatory domains II and IV, respectively (20 -23).
Nitric oxide (NO) is an endogenously synthesized free radical species that has been demonstrated to modulate a variety of cellular and physiological processes. Elevated levels of NO, as are generated by macrophages at sites of inflammation, infection, or during septic shock can potentially lead to nitrosative stress. The effects of nitrosative stress relate to the ability of NO and NO-derived species to react with biological molecules and thereby alter normal cellular functions (24). Alterations in gene expression have been shown to occur in cells subjected to nitrosative stress, because of both direct and indirect effects on various signaling pathways and transcription factors (25,26).
In this study we investigate the effect of NO on NF-B activation and MAP kinase signaling pathways, and demonstrate both time-and concentration-dependent effects. Furthermore, we correlate the effects of NO on signal transduction pathways to the transcriptional expression of two LPS-inducible genes, IFN-␤ and IB-␣. In conclusion, we propose a model for the actions of NO in enhancing and propagating pathological inflammatory conditions and mediating immune responses.
Protein Extraction-Following treatment with experimental agents, cells were washed with cold phosphate-buffered saline, scraped, and collected by centrifugation. For total proteins, cell pellets were resuspended in lysis buffer (10 mM Tris, pH 7.05, 50 mM NaCl, 30 mM sodium pyrophosphate, 2 mM EDTA, 50 mM NaF, 1% (v/v) Triton X-100; 1 mM phenylmethylsulfonyl fluoride, 1 g/ml aprotonin, 1 g/ml leupeptin, 1 g/ml pepstatin A). Samples were incubated on ice for 15 min then centrifuged at 14,000 ϫ g for 15 min at 4°C to remove debris, and supernatant was stored at Ϫ80°C. Protein concentrations were determined by the Bio-Rad protein assay, and samples were diluted to 2 g/l. Nuclear extracts were prepared as previously described, with slight modifications. Briefly, fresh cell pellets were resuspended in 800 l of ice-cold buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride) and 50 l of Igepal CA-630 was added. Nuclei were pelleted by centrifugation at 1000 ϫ g for 5 min at 4°C, and washed twice with buffer A. Nuclear proteins were extracted by addition of buffer B (10 mM HEPES, pH 7.9, 420 mM NaCl, 1.5 mM MgCl 2 , 0.1 mM EDTA, 0.1 mM EGTA, 25% glycerol, 0.5 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride), and centrifuged at 14,000 ϫ g for 10 min at 4°C to remove debris. Nuclear protein concentrations were determined by the Bio-Rad protein assay, and samples were diluted to 1 g/l.
Protein Immunoblots-An equal amount of protein per sample (as indicated in figure legends) was resolved by polyacrylamide gel electrophoresis, and electroblotted onto a 0.2-m nitrocellulose membrane using a Mini Trans-Blot apparatus (Bio-Rad). Membranes were incubated with blocking buffer (20 mM Tris, pH 7.6, 140 mM NaCl, 0.05% Tween 20, 5% nonfat dry milk) prior to incubation with primary antibody diluted in blocking buffer. After washing 3 times in Tris-buffered saline containing 0.1% Tween 20, membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody. Membranes were subsequently washed 3 times in Tris-buffered saline containing 0.1% Tween 20, and a chemiluminescence-based detection with luminol reagent was performed according to the manufacturer's protocol (Santa Cruz Biotechnology, Inc). Antibodies were obtained from the following sources: anti-p38, anti-phospho-p38, anti-JNK, antiphospho-JNK, and anti-IB-␣ from Cell Signaling Technology; anti-p50/p105, anti-p65, anti-actin, and all secondary antibodies were purchased from Santa Cruz Biotechnology, Inc.
Ribonuclease Protection Assay-Following treatment with experimental agents, cells were washed with cold phosphate-buffered saline, harvested using a cell scraper and collected by centrifugation. Total RNA was extracted using the RNeasy Mini kit (Qiagen). RNA was quantified by absorbance at 260 and 280 nm. 15 g of RNA was dried using a Speed-Vac (Savant) and stored at Ϫ80°C. Radiolabeled antisense RNA was synthesized using the Riboprobe transcription kit (Promega) and [␣-32 P]CTP, 800 Ci/mmol (ICN). The antisense templates for murine IFN-␤ and IB-␣ were constructed using a reverse transcriptase-PCR method. Briefly, total RNA collected from LPS-treated RAW 264.7 macrophages was reverse transcribed using the Super-Script preamplification system (Invitrogen). First strand cDNA was subsequently amplified by PCR using the puReTaq Ready-To-Go PCR system (Amersham Biosciences). For IFN-␤, the primers 5Ј-AAACAA-TTTCTCCAGCACTG-3Ј and 5Ј-ATTCTGAGGCATCAACTGAC-3Ј were used, and for IB-␣ the primers 5Ј-TGGCCTTCCTCAACTTCC-3Ј and 5Ј-CTGCGTCAAGACTGCTACAC-3Ј were used. The resulting PCR products were annealed into the pGEM-T Easy vector (Promega), amplified in competent E. coli, and linearized using the restriction enzymes NcoI and SpeI, respectively. The probe for IFN-␤ yields a protected fragment of 312 nucleotides, and the probe for IB-␣ yields a protected fragment of 194 nucleotides. The template for murine ␤-actin was purchased from Ambion and yields a protected fragment of 245 nucleotides. Ribonuclease protection assay was performed using the RPA-III kit (Ambion), but substituting RNase ONE (Promega) for the supplied RNases. Protected fragments were resolved using a 5% polyacrylamide:bisacrylamide (19:1) 25 mM Tris borate, 2.5 mM EDTA, 8 M urea gel. Gels were dried, and exposed to both autoradiographic film and a PhosphorImager cassette (Amersham Biosciences). The radiographic intensities of individual bands on the gels were determined using the program ImageQuant (Amersham Biosciences).

Macrophages Treated with Nitric Oxide Exhibit LPS-independent Phosphorylation of p38 and JNK MAP Kinases-
Treatment of RAW 264.7 macrophages LPS (100 ng/ml) induced a rapid phosphorylation of p38 MAP kinase on amino acid residues threonine 180 (Thr-180) and tyrosine 182 (Tyr-182). Phosphorylation of JNK (p46 and p54) was also induced on amino acid residues threonine 183 (Thr-183) and tyrosine 185 (Tyr-185) (Fig. 1). Nonstimulated macrophages did not have detectable levels of phosphorylated p38 or JNK. At 15 min after LPS addition, phosphorylation of both p38 and JNK was noticeably up-regulated. However, by 45 min levels of phospho-p38 and phospho-JNK had decreased significantly, and by 75 min had returned to an almost undetectable level, as in nonstimulated cells.
Simultaneous treatment of macrophages with LPS and the NO donor, DEA/NO, significantly enhanced phosphorylation of both p38 and JNK. In contrast to macrophages treated with LPS alone, when treated with LPS plus DEA/NO, the levels of phosphorylated p38 and JNK remained elevated at the 45-and 75-min time points after addition. This is despite our calculations that the NO donor was fully degraded at these time points, because of its relatively short half-life (ϳ2 min at 37°C, pH 7.4).
The effect of adding DEA/NO alone on p38 and JNK phosphorylation was also investigated. DEA/NO was capable of inducing a rapid and sustained phosphorylation of both MAP kinases, which was significantly stronger and of greater duration than in cells treated with LPS alone. As a control, the products of DEA/NO decomposition were tested for their ability to activate p38 and JNK phosphorylation. In contrast to freshly prepared DEA/NO, decomposed DEA/NO (obtained by allowing DEA/NO to decay in 0.1 M Tris, pH 7.4, at 25°C for several days), which contains mostly nitrite and diethylamine, had no observable effect on either p38 or JNK phosphorylation.
Nitric Oxide Induces a Sustained Degradation of IB-␣ in LPS-stimulated Macrophages-MAP kinase pathways have been demonstrated to contribute to the activation of NF-B. Therefore, the effect of DEA/NO on the degradation of the NF-B inhibitory protein, IB-␣, was investigated. Accordingly, RAW macrophages were treated with LPS (100 ng/ml), DEA/NO (1 mM), or LPS plus DEA/NO for various amounts of time, and total protein samples were analyzed by immunoblot for IB-␣ expression. Treatment of macrophages with LPS alone induced a rapid degradation of IB-␣ (Fig. 2). A total loss of detectable IB-␣ protein was observed 15 min after LPS addition, but by 45 min the levels of IB-␣ had recovered to the basal level. In contrast, when macrophages were treated simultaneously with a combination of LPS (100 ng/ml) and DEA/NO (1 mM), IB-␣ levels remained low at 45 and 75 min after treatment. The ability of DEA/NO to inhibit the restoration of IB-␣ levels in LPS-stimulated macrophages was both concentration-and time-dependent. Higher concentrations of DEA/NO (100 M to 1 mM) were most effective at inhibiting IB-␣ levels, and at maintaining this inhibition at later time points (Fig. 3). Because DEA/NO alone was capable of inducing p38 and JNK phosphorylation, it was hypothesized that DEA/NO might also induce IB-␣ degradation in the absence of LPS. However, IB-␣ levels in cells not treated with LPS were unaffected by the addition of DEA/NO, indicating that NO alone and the resulting activation of p38 and JNK pathways is insufficient to cause NF-B activation (data not shown).
Nitric Oxide Stimulates Nuclear Accumulation of p50 and p65 and Enhances p105 Turnover-Activation of the transcription factor NF-B is regulated by the degradation of the inhibitor, IB, and the resulting translocation of the p50/p65 heterodimer into the nucleus. Nuclear p50/p65 then mediates the transcription of target genes. Because DEA/NO enhanced the magnitude and duration of LPS-induced IB-␣ degradation, we decided to examine the effect of DEA/NO on LPS-induced NF-B translocation. A modest level of NF-B exists in the nucleus of nonstimulated RAW 264.7 macrophages. When treated with LPS (100 ng/ml) for 75 min, the nuclear levels of p50 and p65 were moderately increased. However, when treated with a combination of LPS and DEA/NO (100 M to 1 mM), the nuclear levels of p50 and p65 increased in a concentration-dependent manner (Fig. 4).
The activity of NF-B in the nucleus is also regulated by the Nuclear extracts were made and 10 g of protein was resolved by polyacrylamide gel electrophoresis. Nitrocellulose blots were probed with antibodies specific for p50/p105, p65, or actin, which was used as a loading control. The immunoblots shown are representative of four individual experiments.
inhibitory actions of IB, which has been reported to enter the nucleus and down-regulate NF-B-mediated gene expression (28). Therefore, the effect of LPS and DEA/NO on nuclear IB-␣ expression was also investigated. Whereas nuclear IB-␣ expression by immunoblot analysis could not be detected, significant levels of the p50 precursor protein, p105 were observed. Like IB, p105 is able to repress NF-B through its inhibitory associations with the transcription factor, masking the nuclear translocation and DNA-binding domains (29). LPS alone did not cause a significant reduction in p105 levels at 45 min following treatment (Fig. 4). However, simultaneous addition of LPS and DEA/NO induced a marked, concentration-dependent decrease in nuclear p105 expression, suggesting that less nuclear NF-B is in an inactive state, and able to promote the transcription of target genes.
Other investigators have reported that NO may inhibit DNA binding of NF-B because of the formation of an S-nitrosothiol on residue Cys-62 of the p50 subunit. Therefore, nuclear protein extracts from DEA/NO-treated macrophages were tested for their ability to bind to a B oligonucleotide. As determined by EMSA, a dramatic increase in NF-B binding was seen at higher DEA/NO concentrations (Fig. 5), consistent with the enhanced p50 and p65 levels in the nucleus (Fig. 4). Nuclear extracts from macrophages were also treated in vitro with DEA/NO to verify that NO could inhibit the DNA binding of NF-B, which has been reported by other investigators (30,31). Nuclear protein extracts from LPS-induced macrophages, or recombinant human p50 were treated with DEA/NO (1 mM final concentration) and analyzed by EMSA in the presence or absence of DTT (1 mM). DTT was added 1 h subsequent to the NO donor to limit the production of HNO from the reaction of NO with DTT. Consistent with the observations in these earlier reports, the addition of NO to NF-B in the absence of reducing thiols completely inhibited NF-B binding to DNA (data not shown). When DTT was added to the reaction after DEA/NO, the inhibition was reversed, and the full level of DNA binding was restored. The results of this experiment are consistent with results from the exposure of NO to cultured cells (Fig. 5). Thus, the reason that the inhibition of NF-B was not observed when cultured cells were treated with DEA/NO is likely because of the high intracellular thiol content, which limits S-nitrosation.
The increase in NF-B activation caused by DEA/NO was significantly greater at higher concentrations (500 M to 1 mM). Therefore, we hypothesized that the effect on NF-B was related to nitrosation reactions associated with the generation of NO 2 and N 2 O 3 , which is facilitated by high NO levels. To examine this possibility, the compound carboxy-PTIO was used. In combination with NO added in the form of DEA/NO, carboxy-PTIO can be utilized to generate NO 2 and N 2 O 3 (32). Thus, carboxy-PTIO enhances the nitrosative reactions caused by the addition of DEA/NO. Accordingly, the addition of DEA/NO (1 mM) alone caused a significant increase in NF-B binding, but the simultaneous addition of DEA/NO and carboxy-PTIO (100 M) caused a further increase in NF-B (Fig.  6). Therefore, we speculate that the biochemical mechanism by which NO increases NF-B activation is likely correlated with a nitrosation event.
Nitric Oxide Enhances the Transcriptional Expression of IFN-␤ and IB-␣-NF-B and the MAP kinase-activated transcription factors c-Jun and activating transcription factor-2 are part of an enhanceosome complex that is responsible for the induction of IFN-␤ in LPS-stimulated or virus-challenged cells. Because DEA/NO enhanced NF-B activation in LPS-treated macrophages and stimulated phosphorylation of p38 and JNK MAP kinases, we decided to investigate the effect of DEA/NO on IFN-␤ gene expression.
Addition of LPS (100 ng/ml) induced a modest expression of IFN-␤ mRNA, measured 3 h later (Fig. 7). Simultaneous treatment with LPS and DEA/NO (100 M to 1 mM) caused a dramatic concentration-dependent increase in IFN-␤ mRNA levels. To examine the contribution of NO-derived nitrosating species (NO 2  Nuclear extracts were made and 5 g of nuclear protein was assayed by electrophoretic mobility shift assay for binding to a consensus B oligonucleotide. Preincubation with unlabeled, cold oligo (C) significantly attenuated binding. Results are representative of five independent experiments. marked increase in IFN-␤ mRNA levels compared with treatment with DEA/NO alone (Fig. 7). This data supports the hypothesis that the stimulatory effect of DEA/NO correlates with the nitrosation of some biochemical species, which promotes the enhanced activation of NF-B and transcription of NF-B regulated genes.
The expression of IB-␣ is also mediated by NF-B, evidenced by the presence of B regulatory elements in its promoter region (33,34). Therefore, the effect of DEA/NO on LPS-stimulated IB-␣ message expression was also examined. Similar to the effect on IFN-␤, the addition of LPS (100 ng/ml) induced a modest expression of IB-␣ mRNA (Fig. 8). Furthermore, simultaneous treatment with LPS and DEA/NO (100 M to 1 mM) caused a dramatic concentrationdependent increase in IB-␣ message expression. However, the magnitude of the effect was not as substantial as the increase in IFN-␤ expression caused by DEA/NO. We currently speculate that the reason IFN-␤ expression is more enhanced by DEA/NO than IB-␣ is because of the additional contribution of MAP kinase-stimulated transcription factors in the IFN-␤ promoter.
The NO donor, DEA/NO, decomposes spontaneously at physiological pH to NO and other products. To confirm that our observations were because of an effect of NO, and not other decomposition products, an additional NO donor was compared for its effects on IFN-␤ and IB-␣ mRNA expression. Thus, the NO donor, MAHMA/NO (500 M, 1 mM) was added to macrophages in combination with LPS (100 ng/ml), and total RNA was extracted 3 h later. RNA was then analyzed by ribonuclease protection assay for the levels of IFN-␤ and IB-␣ transcripts. The addition of MAHMA/NO caused a similar, concentrationdependent enhancement in the expression of IFN-␤ and IB-␣ (Fig. 9). Moreover, the magnitude of the increase in mRNA expression was comparable with that caused by DEA/NO under the same range of concentrations.
We also examined the possibility that an increase in mRNA stability was accounting for the observed elevation in IFN-␤ message levels. However, the addition of actinomycin D (10 g/ml) to inhibit mRNA transcription indicated no differences in IFN-␤ mRNA lifetime between LPS-treated and LPS plus DEA/NO-treated cells (Fig. 10). This data implies that the enhancement of IFN-␤ mRNA levels caused by DEA/NO is not because of a change in message stability, but rather to an increase in the efficiency of transcription.

DISCUSSION
NO is an endogenous free radical species that is both a signaling molecule and a cytotoxic agent (35,36). The function of NO in the immune system is principally to facilitate the killing of invading microorganisms. However, elevated levels of NO can also contribute to various pathological conditions, such as rheumatoid and osteoarthritis, and inflammatory bowel disease (37)(38)(39). In this report we illustrate a mechanism for the enhanced expression of genes by NO because of effects on MAP kinase signaling pathways and NF-B activation. We also provide evidence that NO stimulates the transcriptional expression of the gene encoding the proinflammatory cytokine, IFN-␤.
MAP kinases function in nearly all aspects of the immune response, including the initiation of innate immunity, the activation of adaptive immunity, and in the apoptosis of immune cells (40). In the initiation of a macrophage response to inflammation or infection, MAP kinase-activated transcription factors are critical in triggering the expression of genes encoding cytokines, cell adhesion proteins, and enzymes that mediate the cytotoxic response, including iNOS (41-45). The three main groups of MAP kinases found in mammalian cells include extracellular signal-regulated kinases, p38 MAP kinases, and JNKs. The p38 and JNK pathways are also referred to as the stress-activated MAP kinase pathways because of their more potent activation by stimuli such as inflammatory cytokines and environmental stress (46).
In this report we provide evidence for the activation of p38 MAP kinase and JNK by the NO donor, DEA/NO. The role of NO in regulating MAP kinases has been previously examined in various cell types and iNOS(Ϫ/Ϫ) knockout mice. For example, treatment of Jurkat human T lymphocytes with genuine NO or the iron-nitrosyl compound, sodium nitroprusside (SNP, 0.1-1000 M) was shown to rapidly activate p38 and JNK (47). Adding SNP (1 mM) to RAW 264.7 macrophages also stimulated p38 and JNK MAP kinases (48). Activation of p38 MAP kinase has been reported to mediate the pro-apoptotic effects of NO in several cell types, including human PC-12 cells, rat neural progenitor cells, and rabbit articular chondrocytes (49 -51).
Activation of JNK has been demonstrated in HEK293 cells treated with SNP (500 M), as well as in response to nNOS activation with the ionophore, A23187 (20 M) (52). Also, bovine chondrocytes exhibited an increase in JNK activity when treated with the nitrosothiol, S-nitroso-N-acetylpenicillamine (100 M) (53). In vivo work using iNOS(Ϫ/Ϫ) knockout mice has demonstrated a physiological role for NO in promoting MAP kinase activation. Accordingly, following the induction of septic shock, T-cells and macrophages from iNOS knockouts showed a significant reduction in phospho-p38 levels relative to their wild-type counterparts (54). However, other findings suggest differently, that NO may suppress MAP kinase activation. Accordingly, the nitrosothiol species S-nitroso-N-acetylpenicillamine (100 M) and S-nitrosoglutathione (100 M) were demonstrated to selectively inhibit JNK activity through S-nitrosation of cysteine 116 (55,56). This suggests that NO may have multiple regulatory roles on MAP kinase activities, depending on cell type, donor species, and concentration.
The transcription factor NF-B has a critical role in the immune system, regulating both innate and adaptive responses (57,58). The activation of NF-B is associated with increased transcription of a number of genes involved in immune responses, including those encoding adhesion molecules, cytokines, and iNOS. Expression of these proteins by macrophages enhances the migration of inflammatory cells into areas of infection or inflammation, and contributes to the cytotoxic response.
In this paper we describe a time-and concentration-dependent enhancement of LPS-stimulated NF-B activation by an NO donor. Two mechanisms for the increase in NF-B activation are presented. NO enhanced and prolonged the degradation of the inhibitory protein IB-␣. The addition of NO also stimulated a decrease in the nuclear levels of the inhibitory protein, p105. The combination of these effects we believe contributed to the enhanced nuclear translocation of p50/p65, and the increase in DNA-binding activity determined by EMSA. Interestingly, DEA/NO and MAHMA/NO also stimulated an FIG. 10. IFN-␤ mRNA stability is not affected by DEA/NO. RAW 264.7 macrophages were treated with LPS (100 ng/ml), or LPS ϩ DEA/NO (1 mM). After 3 h of activation, actinomycin D (act-D, 10 g/ml) was added. At various times after the addition of act-D, total RNA was extracted and 15 g of RNA was probed for IFN-␤ and ␤-actin (loading control) by ribonuclease protection assay. Relative values for IFN-␤ mRNA levels normalized to ␤-actin are indicated in the graph below the gel, and were obtained using Phos-phorImager analysis. Results are representative of two independent experiments.
FIG. 11. Model for NO-enhanced induction of IFN-␤ mRNA levels. In cell medium, DEA/NO decomposes spontaneously to release NO, which is freely permeable through biological membranes. NO stimulates phosphorylation of JNK and p38 MAP kinases, and enhances LPS-induced degradation of IB-␣ through unidentified mechanisms. NO also correlates with loss of nuclear p105 levels. Enhanced NF-B and MAP kinase pathways facilitate the assembly of an IFN-␤ enhanceosome, promoting the enhanced expression of the IFN-␤ gene. increase in IB-␣ mRNA expression, likely because of its regulation by NF-B. Therefore, we speculate that the decrease in IB-␣ levels caused by NO donor is not because of an effect of NO on IB-␣ transcription, but rather to a stimulation of IB-␣ protein degradation.
The consequences of NO exposure on NF-B activation have been investigated in several cell types under a variety of conditions. In vitro treatment of recombinant NF-B with nitrosothiols or acidified nitrite has been shown to inhibit DNA binding because of nitrosation of cysteine residue 62 of the p50 subunit (30,31,59,60). Inhibition of NF-B activity by NO has also been attributed to stabilization of IB-␣ in Jurkat T cells and human vascular endothelial cells (61,62).
However, other reports indicate that NO and NO reaction products enhance induction of NF-B. For example, cytokinestimulated nuclear translocation of p50 in rat astroglial cells was enhanced by spermine NONOate (100 M) (63). When also stimulated with IL-1␤ (0.5 ng/ml) and IFN-␥ (50 units/ml), spermine NONOate enhanced both p50 and p65 nuclear translocation. In another study, S-nitrosoglutathione was reported to stimulate NF-B activation by various mechanisms in RAW macrophages. Accordingly, treatment of RAW cells with S-nitrosoglutathione (200 M) alone induced IB-␣ degradation, NF-B DNA binding, and enhanced NF-B-dependent reporter gene expression. The effect of S-nitrosoglutathione on NF-B was also correlated to the expression of cyclooxygenase-2, which was dramatically elevated in comparison to untreated cells (64). In vivo studies using iNOS(Ϫ/Ϫ) knockout mice have also supported a role for NO in promoting NF-B activation. Accordingly, the activation of NF-B caused by induction of hemorrhagic shock was markedly attenuated in the iNOS knockout strain (65).
Interestingly, peroxynitrite, the reaction product of NO and superoxide (O 2 . ), has been shown in several cases to enhance NF-B activation and stimulate the transcriptional expression of NF-B-dependent genes (66 -68). As with the effects of NO on MAP kinase pathways, current data on the inhibition and activation of NF-B suggests multiple modes of regulation by NO, depending on cell type, donor species, and concentration. Accordingly, other investigators have provided evidence for the multifunctional role for NO in the regulation of NF-B (69). These results have indicated a potential physiological role for NO-derived oxidants in promoting the release of proinflammatory cytokines in the human vasculature. They also concur with the findings presented here, that NO enhanced NF-B activation and stimulated IFN-␤ production. Along with interferon regulatory factors, NF-B and c-Jun/ activating transcription factor-2 are responsible for the induction of IFN-␤ in LPS-stimulated or virus-challenged cells (20 -23) (Fig. 11). Our finding that the NO donors, DEA/NO and MAHMA/NO, increase IFN-␤ mRNA levels in LPS-treated macrophages correlates with the enhanced activation of NF-B and MAP kinase signaling cascades. The biochemical mechanism underlying the effects of NO donors reported here remain to be determined. However, the data agree with earlier studies in Jurkat T cells, where SNP, S-nitroso-N-acetylpenicillamine, and genuine NO were shown to enhance p21 ras activity via S-nitrosation of cysteine 118. The NO-induced activation of p21 ras was further correlated to an increase in NF-B and MAP kinase activities (70,71). In conclusion, NF-B and MAP kinase signaling are vital in the regulation of immune functions. Their enhancement by NO, particularly under conditions of sepsis or because of the localized generation of NO by activated macrophages, may promote or maintain an activated or inflammatory state.