- Open Access
Modulation of LPS stimulated NF-kappaB mediated Nitric Oxide production by PKCε and JAK2 in RAW macrophages
© Jones et al; licensee BioMed Central Ltd. 2007
- Received: 16 April 2007
- Accepted: 24 November 2007
- Published: 24 November 2007
Nuclear factor kappa B (NF-κB) has been shown to play an important role in regulating the expression of many genes involved in cell survival, immunity and in the inflammatory processes. NF-κB activation upregulates inducible nitric oxide synthase leading to enhanced nitric oxide production during an inflammatory response. NF-κB activation is regulated by distinct kinase pathways independent of inhibitor of κB kinase (IKK). Here, we examine the role of protein kinase C isoforms and janus activated kinase 2 (JAK2) activation in NF-κB activation and LPS-stimulated NO production.
Murine RAW 264.7 macrophages were treated with lipopolysaccharide (LPS), Phorbol 12-myristate 13-acetate (PMA) and a combination of LPS and PMA in the presence or absence of various inhibitors of PKC isoforms and JAK2. Nuclear translocation of the NF-κB p65 subunit, was assessed by Western blot analysis whilst NO levels were assessed by Greiss assay.
LPS-stimulated NO production was attenuated by PMA whilst PMA alone did not affect NO release. These effects were associated with changes in p65 nuclear translocation. The PKCα, β, γ, δ and ζ inhibitor Gö 6983 (Go) had no effect on LPS-induced NO release. In contrast, Bisindolymalemide I (Bis), a PKC α, βI, βII, γ, δ and ε isoform inhibitors completely inhibited LPS-stimulated NO production without affecting p65 nuclear translocation. Furthermore, a partial inhibitory effect on LPS-induced NO release was seen with the JAK2 inhibitor AG-490 and the p38 MAPK inhibitor SB 203850.
The results further define the role of NF-κB in LPS stimulated NO production in RAW macrophages. The data support a function for PKCε, JAK2 and p38 MAPK in NF-κB activation following p65 nuclear import.
- Nitric Oxide
- NOS2 Expression
- Greiss Assay
Increasing emphasis is currently placed on the role of the innate immune system in inflammatory responses, in particular those involving macrophages. As in other cells, the transcription factor, NF-κB plays a pivotal role in changes in gene expression during such inflammatory responses. A range of inflammatory stimuli, including endotoxin [1, 2] and cytokines , produce activation and nuclear translocation of NFκB following rapid degradation and release of IκB.
One of the genes upregulated by NF-κB during an inflammatory response is the inducible nitric oxide synthase (NOS2), that produces nitric oxide (NO), a highly reactive free radical with important second messenger functions involving the mediation of inflammatory events . Increased expression of NOS2 and concomitant NO levels have been reported in several inflammatory diseases, such as Crohn's disease , asthma  and rheumatoid arthritis . The NOS2 gene promoter contains twenty two putative transcription factor binding elements , however, so far only the NF-κB responsive κB element  and an interferon-γ-activated site (GAS)  have been shown to enhance NOS2 expression.
Macrophages are the primary producers of NO in vivo and one of the dominant cell types to display NF-κB activation in inflammatory diseases . Lipopolysaccharide (LPS) stimulates NO production in macrophages. The induction of NOS2 protein expression in response to stimulation with LPS involves the Janus kinase (JAK) family of protein kinases . Furthermore, both protein kinase C (PKC) and Janus Kinase2 (JAK2) [12–14] have been implicated in NF-κB activation. However, although nine isoforms of PKC have been identified in macrophages  it is unknown which of these are involved in NF-κB activation.
PKC activation has been identified as an early response in LPS-stimulated macrophages  and is essential for the up-regulation of NO production [16, 17] However, the function of PKC isoforms involved in upregulation of NO production remains to be determined. Thus PMA, a direct activator of the PKC family of kinases, was used to investigate the role of PKC in LPS-stimulated NO production and NF-κB activation in RAW cells. PMA has been shown to induce a PKC mediated proteasomal-independent pathway of NF-κB nuclear translocation in human intestinal epithelial cells .
The present study uses pharmacological tools to indicate a role for PKCε in LPS-stimulated NF-κB-mediated NO release in RAW macrophages. We also implicate a role for JAK2 and p38 MAPK on these effects.
RAW 264.7 cells (ECACC, Salisbury, UK) were maintained in 25 cm2 flasks in DMEM medium supplemented with 2 mM L-glutamine and 10% v/v FCS, without antibiotics, at 37°C in a humidified atmosphere of 95% air and 5% CO2. For Western blotting, cells were grown in 25 cm2 flasks, whilst for the measurement of NO the cells were grown to 95% confluence in 96-well plates and stimulation carried out within these plates. Cells were stimulated by replacing the culture medium with medium containing LPS, LPS with phorbol-12-myristate-13-acetate (PMA) or PMA alone in the presence or absence of various inhibitors.
Inhibitors used were: the PKC inhibitors Gö 6983 (Go) and Bisindolymalemide I (Bis); the JAK2 inhibitor AG-490; the p38 MAP kinase inhibitor SB 203580 (Calbiochem, Nottingham, UK). Bis shows high selectivity for PKC α, βI, βII, γ, δ and ε isoforms at 20 μm  whilst Go inhibits PKC α, β, γ, δ and ζ isoforms at 10 μm . AG-490 was used at 10 mM, a concentration previously shown to inhibit JAK2 , and SB 204580 at 10 mM, a concentration previously shown to inhibit the p68 MAP kinase family . For the purpose of specific inhibition of PKC translocation, the following MALY-TAT linked peptides (kindly supplied by Dr M. Lindsay, AstraZeneca, Charnwood, UK) were used: MALYO1 (TAT- RFARKGALRQKNHEVK), MALY1O (TAT-EAVSLKPT), MALY II (TAT-LSETKPAV0) at concentrations previously shown to inhibit translocation of PKC isoforms . For Western blotting cells were incubated for 0, 1, 2, 3 or 5 hours, whilst for the NO assay, cells were incubated for 24 hours.
Assessment of NF-κB-p65 nuclear translocation by Western blot analysis
RAW 264.7 cells were harvested in ice cold PBS after stimulation with LPS from 0 to 5 h. Cells were then lysed in 70 μl of buffer A (10 mM HEPES pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.25% v/v noident P-40, 0.5 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonyl fluoride (PMSF) in de-ionised water (dH2O) for 20 min on ice, to yield the cytoplasmic cellular fraction, as described previously . The samples were microfuged at 12,000 g for 15 sec to pellet the unlysed nuclei and the supernatant (cytoplasmic fraction) was collected. The nuclei were lysed in 15 μl of buffer B (20 mM HEPES pH 7.9, 1.5 mM MgCl2, 0.42 M NaCl, 0.5 mM DTT, 25% v/v Glycerol, 0.5 mM PMSF in diH2O) for 20 min on ice, and microfuged or centrifuged at 12,000 g for 60 sec to pellet the cellular debris. The supernatant (nuclear fraction) was collected and 60 μl of buffer C (20 mM HEPES pH 7.9, 50 mM KCl, 0.5 mM DTT, 0.2 mM EDTA, 0.5 mM PMSF) was added to it. At this stage the protein concentration of the samples was assessed by BioRad protein assay ™ (Biorad, UK).
Samples (10 μg) were separated by 10% SDS-PAGE and proteins transferred to a nitrocellulose membrane (Amersham-Pharmacia, Amersham, UK) by electroblotting. Equal protein loading was confirmed by Ponceau S staining of the membrane. Non-specific protein binding was blocked by incubation of the membrane in PBS-T + 1% w/v milk overnight at 4°C. Membranes were then washed twice in PBS-T for 5 min before incubation for 1 hour at room temperature (RT) with rabbit anti-p65 antibody (1:4000, Santa Cruz, Wembley, UK). Membranes were then washed twice in phosphate buffered saline (pH 7.4) containing 0.05% v/v Tween 20 (PBS-T) and 1% w/v milk for 5 min followed by an hour incubation at room temperature (RT) with goat anti-rabbit HRP conjugate (1:4000, Dako, UK). All antibodies incubations were carried out in PBS-T containing 1% w/v milk. Membranes were washed three times for 5 min in PBS-T before incubation with ECL substrate (Amersham-Pharmacia, UK), followed by exposure to an autoradiographic film and subsequent semi-quantification of band intensity by densitometry (UVP Ltd, Cambridge, UK).
Nitrite determination by Greiss assay
NO levels were assessed by nitrite quantification as described previously . Briefly, 90 μl of sample (cell culture medium) was incubated for 5 min in dark at RT with 90 μl of suphanilamide (1% w/v in 4 M HCl). 90 μl of napthylethylenediamine (1% w/v in dH2O) was then added and a further 5 min incubation was carried out in dark at RT. Absorbance was read at 540 nm.
All reagents were purchased from Sigma (Poole, UK) unless otherwise stated above.
Data are reported as mean ± SEM. Statistical analysis was performed in Prism 5 (Graph Pad Software, Inc. San Diego, USA) using one way analysis of variance (ANOVA) followed by Tukey's Multiple Comparison Test (TMCT) when ANOVA indicated a statistical significance existed.
The effect of LPS and PMA on RAW cell NO production
LPS stimulation induced NO production with nitrite levels peaking at 33 μM (p < 0.001, Fig. 1), in agreement with the results of others [26, 27]. In contrast, PMA alone (0.5–500 ng/ml) had no effect on NO production but significantly attenuated LPS-induced NO production by ~50% (Fig. 1) even at concentrations (50 ng/ml) previously shown to activate NF-κB.
The effect of LPS on NF-κB activation
The role of PKC in LPS stimulated RAW cell NO production
Nitrite (μM); mean ± SEM
Concentration of peptide alone
1 μg/ml LPS with stated concentration of peptides below
The effect of AG-490 on LPS induced RAW cell NO production
The effect of SB 203580-induced inhibition of p38 on LPS induced RAW cell NO production
This study investigated the effect of distinct kinase pathways on their ability to modulate NF-κB activation and thereby modify LPS stimulated NO production in RAW macrophages. Previous findings have been extended, with NF-κB-mediated NO production stimulated by LPS is shown to be multifactorial in nature, involving the co-ordinated activation of PKCε, JAK2 and p38 MAPK. The differential effects of the PKC inhibitors Go and Bis suggested that PKCε was involved in NO release.
Under unstimulated conditions, p65 is restricted to the cytoplasm by a set of inhibitory proteins and, upon stimulation, translocated to the nucleus. This stimulation can be modulated by phosphorylation of p65 at serine residues. The degree of activation by NF-κB is thus likely to result from a combination of p65 nuclear translocation and post-translational modifications of p65 .
It is evident from many studies that LPS-stimulated NO release from RAW macrophages is NF-κB dependent . In the present study, although PMA enhanced the amount of p65 nuclear translocation, it also decreased the period over which LPS was able to maintain NF-κB nuclear translocation and this may be linked to the reduction in NO release.
Inhibition of PKCε by Bis had no effect on nuclear translocation in our study confirming a previous report in LPS-stimulated blood monocytes . This data, in conjunction with the data showing a lack of effect of a TAT-linked MALY inhibitor, indicates that PKC is not involved in the nuclear translocation or DNA-binding of NF-kB. Thus, PKCε probably acts on nuclear NF-κB to either affect its nuclear retention or more likely to affect p65 transcriptional activity through a posttranslational modification event leading to differential recruitment or activation of transcriptional co-activators. Indeed, a PKC phosphorylation site exists on the p65 subunit and such phosphorylation is known to increase the transactivation potential of NF-κB without affecting its DNA binding or nuclear translocation [38, 39]. Furthermore, the data presented here suggests that the effect of PMA on LPS-stimulated NO release is not through a PKC-mediated effect but that PMA induces additional pathways that regulate LPS-induced NF-κB activation and NO production. Thus, altered PKC activity may also impinge upon the NF-κB functional response either by affecting co-factor or histone phosphorylation .
A role for JAK2 in LPS and IFN stimulated NO production in RAW cells has been described previously . However, JAK2 was hypothesised to work solely through STAT1 activation and be activated by IFNγ. There is currently growing evidence for cross talk between the JAK2 and the NF-κB signalling pathway  and also the JNK pathway indirectly through an effect on PI3K [43, 44]. JAK2 has been demonstrated to phosphorylate IkB thereby facilitating NF-κB activation . AG-490 has also been reported to inhibit LPS stimulated NF-κB activation and subsequent NOS2 induction in a skin dendritic cell line .
As with JAK2, the results from the present study suggest that the p38 MAPK protein (MAP) kinase is also involved in NF-κB activation [29, 33] and therefore LPS-induced NO production. Previous reports have shown equivocal data as to the role of p38 MAPK in these events [46, 47]. Although p38 MAPK does not appear to be involved in NO release induced by other agents in RAW cells . The IC50 of inhibition of SB203580 indicates relative selectivity and further analysis of LPS-induced p38 MAPK activation or the use of more selective inhibitors may provide additional evidence for its role effects in these cells and for p38 inhibitors potential in the treatment of LPS activated disease.
This work was funded by the University of Bedfordshire and GlaxoSmithKline (UK). The MALY-TAT linked peptides were kindly supplied by Dr M. Lindsay, AstraZeneca, Charnwood, UK.
- Zhang G, Ghosh S: Toll-like receptor-mediated NF-kappaB activation: a phylogenetically conserved paradigm in innate immunity. J Clin Invest. 2001, 107: 13-9.PubMedPubMed CentralView ArticleGoogle Scholar
- Collins T, Read MA, Neish AS, Whitley MZ, Thanos D, Maniatis T: Review: Transcriptional regulation of endothelial cell adhesion molecules: NF-kappaB and cytokine-inducible enhancers. FASEB. 1995, 9: 899-909.Google Scholar
- Jobin C, Hellerbrand C, Licato LL, Brenner DA, Sartor RB: Mediation by NF-kappaB of cytokine induced expression of intercellular adhesion molecule 1 (ICAM-1) in an intestinal epithelial cell line, a process blocked by proteasome inhibitors. Gut. 1998, 42: 779-787.PubMedPubMed CentralView ArticleGoogle Scholar
- McCafferty DM: Peroxynitrite and inflammatory bowel disease. Gut. 2000, 46: 436-439. 10.1136/gut.46.3.436.PubMedPubMed CentralView ArticleGoogle Scholar
- Boughton-Smith NK, Evans SM, Hawkey CJ, Cole AT, Balsitis M, Whittle BJ, Moncada S: Nitric oxide synthase activity in ulcerative colitis and Crohn's disease. Lancet. 1993, 342: 338-340. 10.1016/0140-6736(93)91476-3.PubMedView ArticleGoogle Scholar
- Kharitinov SA, Barnes JP: Effects of corticosteroids on noninvasive biomarkers of inflammation in asthma and chronic obstructive pulmonary disease. Proc Am Thorac Soc. 2004, 1: 191-199. 10.1513/pats.200402-011MS.View ArticleGoogle Scholar
- Yasuda T, Kakinuma T, Julovi SM, Hiramatsu T, Akiyoshi M, Nakamura T: COOH- terminal heparin-binding fibronectin fragment induces nitric oxide production in rheumatoid cartilage through CD44. Rheumatology. 2004, 43: 1116-1120. 10.1093/rheumatology/keh274.PubMedView ArticleGoogle Scholar
- Xie Q-W, Kashiwabara Y, Nathan C: Role of transcription factor NF-kappaB/Rel in induction of nitric oxide synthase. J Biol Chem. 1994, 269: 4705-4708.PubMedGoogle Scholar
- Kim Y-M, Lee S-B, Yi K-Y, Palk S-G: Upstream NF-kappaB site is required for the maximal expression of mouse inducible nitric oxide synthase gene in interferon-g plus lipopolysaccharide-induced RAW 264.7 macrophages. Biochem Biophys Res Comm. 1997, 236: 655-660. 10.1006/bbrc.1997.7031.PubMedView ArticleGoogle Scholar
- MacMicking J, Xie Q-W, Nathan C: Nitric oxide and macrophage function. Annu Rev Immunol. 1997, 15: 323-350. 10.1146/annurev.immunol.15.1.323.PubMedView ArticleGoogle Scholar
- Ihle JN: Cytokine receptor signalling. Nature. 1995, 377: 591-594. 10.1038/377591a0.PubMedView ArticleGoogle Scholar
- Steffan NM, Bren GD, Frantz B, Tocci MJ, O'Neill EA, Paya CV: Regulation of IκB phosphorylation by PKC and Ca2+ dependent signal transduction pathways. J Immunol. 1995, 155: 4685-4691.PubMedGoogle Scholar
- Cataisson C, Pearson AJ, Torgerson S, Nedospasov SA, Yuspa ST: Protein kinase Cα-mediated chemotaxis of neutrophils requires NF-kB activity but is independent o TNFα signalling in moiuse skin in vivo. J Immunol. 2005, 174: 1686-1692.PubMedView ArticleGoogle Scholar
- Digicaylioglu M, Lipton SA: Erythropoietin-mediated neuroprotection involves cross-talk between JAK2 and NFκB signalling cascades. Nature. 2001, 412: 641-647. 10.1038/35088074.PubMedView ArticleGoogle Scholar
- Lin W-W, Chen BC: Distinct isoforms mediate the activation of cPLA2 and adenylyl cyclase by phorbal ester in RAW 264.7 macrophages. Brit J Pharmocol. 1998, 125: 1601-1609. 10.1038/sj.bjp.0702219.View ArticleGoogle Scholar
- Paul A, Pendreigh RH, Plevin R: Protein kinase C and tyrosine kinase pathways regulate lipopolysacchride-induced nitric oxide synthase activity in RAW 264.7 macrophages. Brit J Pharmacol. 1995, 114: 482-488.View ArticleGoogle Scholar
- Severn S, Wakelam MJO, Liew FY: The role of PKC in the induction of nitric oxide synthase by murine macrophages. Biochem Biophys Res Comm. 1993, 188: 199-1002.Google Scholar
- Sun Y, Oberley L: Redox regulation of transcriptional activators. Free Radic Biol Med. 1996, 21: 335-348. 10.1016/0891-5849(96)00109-8.PubMedView ArticleGoogle Scholar
- Ku WC, Cheng AJ, Wang TC: Inhibition of telomerase activity by PKC inhibitors in human nasopharyngeal cancer cells in culture. Biochem Biophys Res Comm. 1997, 241: 730-6. 10.1006/bbrc.1997.7874.PubMedView ArticleGoogle Scholar
- Gschwendt M, Dieterich S, Rennecke J, Kittstein W, Mueller HJ, Johannes FJ: Inhibition of protein kinase C mu by various inhibitors. Differentiation from protein kinase c isoenzymes. FEBS Letts. 1996, 392: 77-80. 10.1016/0014-5793(96)00785-5.View ArticleGoogle Scholar
- Levitzki A: Tyrphostins – potential antiproliferative agents and novel molecular tools. Biochem Pharmacol. 1990, 40: 913-918. 10.1016/0006-2952(90)90474-Y.PubMedView ArticleGoogle Scholar
- Lee JC, Laydon JT, McDonnell PC, Gallagher TF, Kumar S, Green D, McNulty D, Blumenthal MJ, Heys JR, Landvatter SW: A protein kinase involved in the regulation of inflammatory cytokine biosynthesis. Nature. 1994, 372: 739-746. 10.1038/372739a0.PubMedView ArticleGoogle Scholar
- Yedovitzky M, Mochly-Rosen D, Johnson JA, Gray MO, Ron D, Abramovitch E, Cerasi E, Nesher R: Translocation inhibitors define specificity of protein kinase C isoenzymes in pancreatic beta-cells. J Biol Chem. 1997, 272: 1417-1420. 10.1074/jbc.272.3.1417.PubMedView ArticleGoogle Scholar
- Osborn L, Kunel S, Nabel GJ: Tumour necrosis factor-α and interleukin-1 stimulate the human immunodeficiency virus enhancer by activation of NF-kappaB. Proc Natl Acad Sci. 1989, 86: 2336-2340. 10.1073/pnas.86.7.2336.PubMedPubMed CentralView ArticleGoogle Scholar
- Tsai SH, Lin-Shiau SY, Lin JK: Suppression of nitric oxide synthase and the down-regulation of the activation of NF-kappaB in macrophages by resveratrol. J Pharmacol. 1999, 126: 673-680.Google Scholar
- Paul A, Bryant C, Lawson MF, Chilvers ER, Plevin R: Dissociation of lipopolysaccharide-mediated induction of nitric oxide synthesis and inhibition of DNA synthesis in RAW 264.7 macrophages and rat aortic smooth muscle cells. Brit J Pharmacol. 1997, 120: 1439-1444. 10.1038/sj.bjp.0701070.View ArticleGoogle Scholar
- Patel R, Attur MG, Dave MN, Kumar S, Lee JC, Abramson SB, Amin AR: Regulation of nitric oxide and prostaglandin E2 production by CSAIDS (SB203580) in murine macrophages and bovine chondrocytes stimulated with LPS. Inflamm Res. 1999, 48: 337-43. 10.1007/s000110050469.PubMedView ArticleGoogle Scholar
- Paul A, Doherty K, Plevin R: Differential regulation by protein kinase C isoforms of nitric oxide synthase induction in RAW 264.7 macrophages and rat aortic smooth muscle cells. Brit J Pharmocol. 1997, 120: 940-946. 10.1038/sj.bjp.0700976.View ArticleGoogle Scholar
- Mayne GC, Murray AW: Evidence that protein kinase C mediates phorbal ester inhibition of calphostin C- and tumor necorsis factor – induced apoptosis in U937 histiocytic lymphoma cells. J Biol Chem. 1998, 273: 24115-24121. 10.1074/jbc.273.37.24115.PubMedView ArticleGoogle Scholar
- Parganas E, Wang D, Stravopodis D, Topham DJ, Jean-Christopher M, Teglund S, Nanin EF, Bodner S, Colamonici OR, Deursen JM, Groisveld G, Ihle JN: Jak2 is essential for signalling through a variety of cytokine receptors. Cell. 1998, 93: 385-395. 10.1016/S0092-8674(00)81167-8.PubMedView ArticleGoogle Scholar
- Kleinert H, Euchenhofer C, Fritz G, Ihrig-Biedert I, Forstermann U: Involvement of protein kinases in the induction of NO synthase II in human DLD-1 cells. Brit J Pharmocol. 1997, 123: 1716-1722. 10.1038/sj.bjp.0701782.View ArticleGoogle Scholar
- Marrero MB, Venema VJ, He H: Inhibition by the Jak/Stat pathway of IFNγ- and LPS-stimulated nitric oxide synthase induction in vascular smooth muscle cells. Biochem Biophys Res Comm. 1998, 252: 508-512. 10.1006/bbrc.1998.9678.PubMedView ArticleGoogle Scholar
- Berghe WV, Plaisance S, Boone E, Bosscher KD, Schmitz ML, Fiers W, Haegeman G: p38 and extracellular signal-regulated kinase mitogen activate protein kinase pathways are required for nuclear factor-B p65 transactivation mediated by tumor necrosis factor. J Biol Chem. 1998, 273: 3285-3290. 10.1074/jbc.273.6.3285.View ArticleGoogle Scholar
- Dorn GWII, Tepe NM, Wu G, Yatani A, Liggett SB: Mechanisms of impaired beta-adrenergic receptor signaling in Gq-mediated cardiac hypertrophy and ventricular dysfunction. Mol Pharmacol. 2000, 57: 278-287.PubMedGoogle Scholar
- Caramori G, Adcock IM, Ito K: Anti-inflammatory inhibitors of IkappaB kinase in asthma and COPD. Curr Opin Investig Drugs. 2004, 5: 1141-7.PubMedGoogle Scholar
- Bae IK, Min HY, Han AR, Seo EK, Lee SK: Suppression of lipopolysaccharide-induced expression of inducible nitric oxide synthase by brazilin in RAW 264.7 macrophage cells. Eur J Pharmacol. 2005, 513: 237-42. 10.1016/j.ejphar.2005.03.011.PubMedView ArticleGoogle Scholar
- Shames BD, Selzman CH, Puliido EJ, Meng X, Meldrum DR, McIntyre RC, Harken AH, Banerjee A: LPS-induced NF-kappaB activation and TNF- release in human monocytes are protein tyrosine kinase dependent and protein kinase C independent. J Surg Re. 1999, 83: 69-74. 10.1006/jsre.1998.5564.View ArticleGoogle Scholar
- Wang D, Westerheide SD, Hanson JL, Baldwin AS: Tumor necrosis factor alpha-induced phosphorylation of RelA/p65 on Ser529 is controlled by casein kinase II. J Biol Chem. 2000, 275: 32592-32597. 10.1074/jbc.M001358200.PubMedView ArticleGoogle Scholar
- Zhong H, SuYang H, Erdjument-Bromage H, Tempst P, Ghosh S: The transcriptional activity of NF-kappaB is regulated by the IkappaB-associated PKAc subunit through a cyclic AMP-independent mechanism. Cell. 1997, 89: 413-24. 10.1016/S0092-8674(00)80222-6.PubMedView ArticleGoogle Scholar
- Edmunds JW, Mahadevan LC: MAP kinases as structural adaptors and enzymatic activators in transcription complexes. J Cell Sci. 2004, 117: 3715-23. 10.1242/jcs.01346.PubMedView ArticleGoogle Scholar
- Delgado M, Ganea D: Inhibition of IFN-gamma-induced janus kinase-1-STAT1 activation in macrophages by vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide. J Immunol. 2000, 165: 3051-7.PubMedView ArticleGoogle Scholar
- Digicaylioglu M, Lipton S: Erthropoietin-mediated neuroprotection involves cross-talk between Jak2 and NF-kB signalling cascades. Nature. 2001, 412: 641-647. 10.1038/35088074.PubMedView ArticleGoogle Scholar
- Okugawa S, Ota Y, Kitazawa T, Nakayama K, Yanagimoto S, Tsukada K, Kawada M, Kimura S: Janus kinase 2 is involved in lipopolysaccharide-induced activation of macrophages. Am J Physiol Cell Physiol. 2003, 285: C399-408.PubMedView ArticleGoogle Scholar
- Alblas J, Honing H, de Lavalette CR, Brown MH, Dijkstra CD, van den Berg TK: Signal regulatory protein alpha ligation induces macrophage nitric oxide production through JAK/STAT- and hosphatidylinositol 3-kinase/Rac1/NAPDH oxidase/H2O2-dependent pathways. Mol Cell Biol. 2005, 25: 7181-92. 10.1128/MCB.25.16.7181-7192.2005.PubMedPubMed CentralView ArticleGoogle Scholar
- Cruz MT, Duarte CB, Goncalo M, Carvalho AP, Lopes MC: LPS induction of IkappaB-alpha degradation and iNOS expression in a skin dendritic cell line is prevented by the Janus kinase 2 inhibitor, Tyrophostin B42. Nitric Oxide. 2001, 5: 53-61. 10.1006/niox.2000.0320.PubMedView ArticleGoogle Scholar
- Paul A, Cuenda A, Bryant CE, Murray J, Chilvers ER, Cohen P, Gould GW, Plevin R: Involvement of mitogen-activated protein kinase homologues in the regulation of lipopolysaccharide-mediated induction of cyclo-oxygenase-2 but not nitric oxide synthase in RAW 264.7 macrophages. Cell Signal. 1999, 11: 491-7. 10.1016/S0898-6568(99)00018-2.PubMedView ArticleGoogle Scholar
- Ajizian SJ, English BK, 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. 1999, 179: 939-44. 10.1086/314659.PubMedView ArticleGoogle Scholar
- Poteser M, Wakabayashi I: Serum albumin induces iNOS expression and NO production in RAW 267.4 macrophages. Br J Pharmacol. 2004, 143: 143-51. 10.1038/sj.bjp.0705897.PubMedPubMed CentralView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.