Vitamin E γ-Tocotrienol Inhibits Cytokine-Stimulated NF-κB Activation by Induction of Anti-Inflammatory A20 via Stress Adaptive Response Due to Modulation of Sphingolipids

NF-kappa B plays a central role in pathogenesis of inflammation and cancer. Many phytochemicals, including gamma-tocotrienol (gamma TE), a natural form of vitamin E, have been shown to inhibit NF-kappa B activation, but the underlying mechanism has not been identified. In this study, we show that gamma TE inhibited cytokine-triggered activation of NF-kappa B and its upstream regulator TGF-beta-activated kinase-1 in murine RAW 264.7 macrophages and primary bone marrow-derived macrophages. In these cells, gamma TE induced upregulation of A20, an inhibitor of NF-kappa B. Knockout of A20 partially diminished gamma TE's anti-NF-kappa B effect, but gamma TE increased another NF-kappa B inhibitor, Cezanne, in A20(-/-) cells. In search of the reason for A20 upregulation, we found that gamma TE treatment increased phosphorylation of translation initiation factor 2, IkB alpha, and JNK, indicating induction of endoplasmic reticulum stress. Liquid chromatography-tandem mass spectrometry analyses revealed that gamma TE modulated sphingolipids, including enhancement of intracellular dihydroceramides, sphingoid bases in de novo synthesis of the sphingolipid pathway. Chemical inhibition of de novo sphingolipid synthesis partially reversed gamma TE's induction of A20 and the anti-NF-kappa B effect. The importance of dihydroceramide increase is further supported by the observation that C8-dihydroceramide mimicked gamma TE in upregulating A20, enhancing endoplasmic reticulum stress, and attenuating TNF-triggered NF-kappa B activation. Our study identifies a novel anti-NF-kappa B mechanism where A20 is induced by stress-induced adaptive response as a result of modulation of sphingolipids, and it demonstrates an immunomodulatory role of dihydrocermides.