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The production and release of inflammatory mediators is regulated by the coordinated activity of kinases and phosphatases. These proteins are known to regulate one another through an unknown mechanism. Previously, we have demonstrated that autocrine release of oxidants regulates macrophage activation in a similar fashion. The purpose of this study is to determine if attenuated oxidant activity by antioxidant exposure can regulate endotoxin-mediated kinase and phosphatase activity. Human promonocytic THP-1 cells were stimulated with lipopolysaccharide. Selected cells were pretreated with α-tocopherol succinate, LY294002, or an AKT inhibitor (1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate). Lipid raft and cellular protein were analyzed for lipid raft toll-like receptor 4 (TLR4) receptor formation and mitogen-activated protein kinase (MAPK) activation. Harvested supernatants were analyzed for tumor necrosis factor (TNF)-α production. Lipopolysaccharide stimulation led to the lipid raft mobilization of TLR4 and heat shock protein 70. This was followed by lipid raft mobilization of SH related complex homology 2 domain-containing inositol-5-phosphate (SHIP), activation of the MAPK, and production of TNF-α. Pretreatment with α-tocopherol succinate did not affect mobilization of TLR4 or heat shock protein 70, but did result in attenuated mobilization of SHIP, activation of the MAPK, and production of TNF-α. In addition, α-tocopherol succinate was associated with increased activation of the counter-regulatory kinase protein kinase B. Pretreatment with LY294002 or 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate reversed the effects of α-tocopherol succinate. Thus, it seems that endotoxin-mediated activation requires the coordinated activity of kinases and phosphatases. Antioxidant exposure in the form of vitamin E seems to attenuate endotoxin-mediated SHIP activation resulting in increased AKT activity, and attenuated MAPK activation and TNF-α production.