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The Journal of Neuroscience, March 1, 1998, 18(5):1633-1641

Extracellular Signal-Regulated Kinase and p38 Subgroups of Mitogen-Activated Protein Kinases Regulate Inducible Nitric Oxide Synthase and Tumor Necrosis Factor- Gene Expression in Endotoxin-Stimulated Primary Glial Cultures

Narayan R. Bhat¹, Peisheng Zhang¹, John C. Lee², and Edward L. Hogan¹

¹ Department of Neurology, Medical University of South Carolina, Charleston, South Carolina 29425, and ² SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406

Tumor necrosis factor- (TNF) and nitric oxide (NO), the product of inducible NO synthase (iNOS), mediate inflammatory and immune responses in the CNS under a variety of neuropathological situations. They are produced mainly by "activated" astrocytes and microglia, the two immune regulatory cells of the CNS. In this study we have examined the regulation of TNF and iNOS gene expression in endotoxin-stimulated primary glial cultures, focusing on the role of mitogen-activated protein (MAP) kinase cascades. The bacterial lipopolysaccharide (LPS) was able to activate extracellular signal-regulated kinase (ERK) and p38 kinase subgroups of MAP kinases in microglia and astrocytes. ERK activation was sensitive to PD98059, the kinase inhibitor that is specific for ERK kinase. The activity of p38 kinase was inhibited by SB203580, a member of the novel class of cytokine suppressive anti-inflammatory drugs (CSAIDs), as revealed by blocked activation of the downstream kinase, MAP kinase-activated protein kinase-2. The treatment of glial cells with either LPS alone (microglia) or a combination of LPS and interferon- (astrocytes) resulted in an induced production of NO and TNF. The two kinase inhibitors, at micromolar concentrations, individually suppressed and, in combination, almost completely blocked glial production of NO and the expression of iNOS and TNF, as determined by Western blot analysis. Reverse transcriptase-PCR analysis showed changes in iNOS mRNA levels that paralleled iNOS protein and NO while indicating a lack of effect of either of the kinase inhibitors on TNF mRNA expression. The results demonstrate key roles for ERK and p38 MAP kinase cascades in the transcriptional and post-transcriptional regulation of iNOS and TNF gene expression in endotoxin-activated glial cells.

Key words: astrocytes; microglia; TNF; inducible nitric oxide synthase; p38 MAP kinase; extracellular signal-regulated kinase

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Copyright © 1998 Society for Neuroscience  0270-6474/98/1851633-09$05.00/0

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