P38 mitogen-activated protein kinase inhibitor SB203580 has a bi-directional effect on iNOS expression and NO production

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Abstract

In the present study, the mediator role of p38 kinase, a member of the mitogen-activated protein kinase (MAPK) family, was studied in lipopolysaccharide-induced inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production in J774 mouse macrophages and T-84 human colon epithelial cells. Two pyridinyl imidazole inhibitors of p38 MAPK, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-imidazole (SB203580) and 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-imidazole (SB202190), stimulated NO production at low drug concentrations, maximal stimulation occurring at 1 μM drug concentration. In contrast, higher concentrations inhibited NO production, which was>90% at 30 μM drug concentration. The bi-directional effect was found in both cell types tested. Negative control compound SB202474, which is structurally related but does not inhibit p38, did not stimulate NO production but inhibited it at 30 μM concentration. A chemically different p38 inhibitor 2-methyl-4-phenyl-5-(4-pyridyl)oxazole (SC68376) had only a stimulatory action on NO production. Western blotting and reverse transcriptase polymerase chain reaction (RT-PCR) analysis of iNOS showed that both stimulatory and inhibitory effects of SB203580 occurred at the level of iNOS expression. SB203580 did not alter lipopolysaccharide-induced NF-κB activation as detected by electrophoretic mobility shift assay (EMSA). The data show that pyridinyl imidazoles SB203580 and SB202190 have a bi-directional effect on NO production through iNOS pathway depending on the drug concentration used. The inhibitory effect was unrelated to inhibition of p38 MAPK, whereas the stimulatory effect is most likely mediated by p38 MAPK dependent mechanism, suggesting a novel mechanism for regulation of iNOS expression, which is common for murine and human cells.

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.

References (36)

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