Superoxide anion contributes to the induction of tumor necrosis factor alpha (TNFα) through activation of the MKK3/6-p38 MAPK cascade in rat microglia

Abstract

Stimulation of rat microglia with lipopolysaccharide (LPS) in vitro induces production of the inflammatory/cytotoxic cytokine tumor necrosis factor alpha (TNFα) along with superoxide anion (O(2)(-)) and nitric oxide (NO). In this study, we investigated the role of O(2)(-) and NO in the induction of TNFα in microglia. The LPS-inducible TNFα was significantly suppressed by pretreatment with the O(2)(-) scavenger N-acetyl cysteine (NAC), but not by the NO scavenger 2-(4-Carboxyphenyl)-4,4,5,5-tetramethyl imidazoline-1-oxyl 3-oxide, suggesting the close association of O(2)(-) with TNFα induction. NAC strongly depressed phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), which is necessary for inducing TNFα in microglia. On the other hand, an O(2)(-) donor, 3-(4-Morpholinyl)sydnonimine (SIN-1), induced TNFα in microglia, and the effects of SIN-1 were completely abolished in the presence of superoxide dismutase. There is little likelihood that the NO produced in SIN-1 degradation induces TNFα in microglia, because TNFα was not induced in microglia exposed to the NO-donor S-nitroso-N-acetyl-dl-penicillamine. Moreover, the addition of SIN-1 to microglia resulted in activation of p38 MAPK and its upstream kinase MKK3/6. Taken together, these results showed that O(2)(-) is an important signaling molecule for activating the MKK3/6-p38 cascade, which is requisite for inducing TNFα in microglia.