EXTRACELLULAR SIGNAL-REGULATED KINASE AND P38 SUBGROUPS OF MITOGEN-ACTIVATED PROTEIN-KINASES REGULATE INDUCIBLE NITRIC-OXIDE SYNTHASE AND TUMOR-NECROSIS-FACTOR-ALPHA GENE-EXPRESSION IN ENDOTOXIN-STIMULATED PRIMARY GLIAL CULTURES

Tumor necrosis factor-alpha (TNF alpha) and nitric oxide (NO), the pro duct 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, th e two immune regulatory cells of the CNS. In this study we have examin ed the regulation of TNF alpha and iNOS gene expression in endotoxin-s timulated primary glial cultures, focusing on the role of mitogen-acti vated 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 S B203580, a member of the novel class of cytokine suppressive anti-infl ammatory drugs (CSAIDs), as revealed by blocked activation of the down stream kinase, MAP kinase-activated protein kinase-2. The treatment of glial cells with either LPS alone (microglia) or a combination of LPS and interferon-gamma (astrocytes) resulted in an induced production o f NO and TNF alpha. The two kinase inhibitors, at micromolar concentra tions, individually suppressed and, in combination, almost completely blocked glial production of NO and the expression of iNOS and TNF alph a, as determined by Western blot analysis. Reverse transcriptase-PCR a nalysis showed changes in iNOS mRNA levels that paralleled iNOS protei n and NO while indicating a lack of effect of either of the kinase inh ibitors on TNF alpha mRNA expression. The results demonstrate key role s for ERK and p38 MAP kinase cascades in the transcriptional and post- transcriptional regulation of iNOS and TNF alpha gene expression in en dotoxin-activated glial cells.