P38 mitogen-activated protein kinase inhibitor SB203580 has a bi-directional effect on iNOS expression and NO production
Introduction
Nitric oxide (NO) serves as a cytotoxic and cytostatic agent and modulates immune response in inflammatory diseases (Moilanen et al., 1999). High amounts of NO are produced from l-arginine by inducible nitric oxide synthase (iNOS), which is expressed in cells in response to various inflammatory cytokines and bacterial products, e.g. lipopolysaccharide (MacMicking et al., 1997).
Mitogen-activated protein kinases (MAPKs) are a family of serine/threonine specific kinases, which together with their upstream activators form distinct but partially overlapping signalling pathways, which are activated by various inflammatory and other stimuli (Su and Karin, 1996). We have recently shown that extracellular signal-regulated kinases p42/p44, which belong to the MAPK family, are involved in the up-regulation of lipopolysaccharide-induced iNOS expression (Lahti et al., 2000). Lipopolysaccharide-induced production of pro-inflammatory cytokines tumor necrosis factor-α and interleukin-1 is known to be controlled by p38, another kinase of the MAPK family (Su and Karin, 1996). Therefore, we wanted to investigate whether p38 MAPK participates also in the regulation of lipopolysaccharide-induced iNOS expression and NO production.
Pyridinyl imidazole SB203580 is a widely used inhibitor of p38 MAP kinase. It inhibits the activity of p38 MAPK at IC50 of ∼0.3–0.6 μM (Cuenda et al., 1995) by binding in ATP site (Young et al., 1997). In the present study, SB203580 was employed as pharmacological tool to investigate the role of p38 MAPK in the lipopolysaccharide-induced NO production in two different cell lines. J774 murine macrophages were chosen because the mechanisms of NO production have been widely studied in this cell line. To see whether the results obtained in J774 cells are applicable to human cells, T-84 human colon epithelial cell line was used. NO production and iNOS expression in T-84 cells has been recently characterised in our laboratory (Lähde et al., 2000).
The results show that pyridinyl imidazole SB203580 has a bi-directional, concentration-dependent effect on lipopolysaccharide-induced iNOS expression and NO production and only the enhancing effect is likely to be related to inhibition of p38.
Section snippets
Materials
Reagents were obtained as follows: 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-imidazole (SB203580) and 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-imidazole (SB202190) (Alexis, Läufelfingen, Switzerland), 4-ethyl-2-(4-methoxyphenyl)-5-(4-pyridyl)-imidazole (SB202474) and 2-methyl-4-phenyl-5-(4-pyridyl)oxazole (SC68376) (Calbiochem, La Jolla, CA, USA), ammonium pyrrolidinedithiocarbamate (PDTC) (Tocris Cookson, Bristol, UK), rabbit polyclonal mouse and human iNOS and
SB203580 and SB202190 have a concentration-dependent bi-directional effect on lipopolysaccharide-induced NO production
We tested the effects of SB203580 and SB202190 on lipopolysaccharide-induced NO production in J774 mouse macrophages and T-84 human colon epithelial cells. Nitrite accumulated in culture medium was used as a measure of NO production. Both compounds had a clear concentration-dependent effect on lipopolysaccharide-induced NO production in both cell lines (Fig. 1). Low concentrations of SB203580 and SB202190 increased NO production and maximal stimulatory effect was achieved at 1 μM drug
Discussion
We have shown in this study that p38 MAPK inhibitors SB203580 and SB202190 have a concentration-dependent bi-directional effect on lipopolysaccharide-induced NO production in murine J774 macrophages and T-84 human colon epithelial cells. Furthermore, we have found that both the stimulatory and inhibitory effects of SB203580 occur at the level of iNOS expression, by a NF-κB-independent mechanism.
Stimulatory effect of p38 MAPK inhibitors SB203580 and SB202190 on lipopolysaccharide-induced NO
Acknowledgements
We thank Mrs. Niina Ikonen for the skillful technical assistance and Mrs. Heli Määttä for secretarial help.
The study was supported by grants from The Academy of Finland, The Medical Research Fund of Tampere University Hospital and Tampere Tuberculosis Foundation.
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