Vitamin E δ-tocotrienol inhibits TNF-α-stimulated NF-κB activation by up-regulation of anti-inflammatory A20 via modulation of sphingolipid including elevation of intracellular dihydroceramides

Nuclear factor-kappa B (NF-kappa B) regulates inflammation and cell survival, and is considered a potential target for anti-inflammatory and anti-cancer therapy. 6-Tocotrienol (STE), a vitamin E form, has been shown to inhibit NF-kappa B, but the mechanism underlying this action is not clear. In the present study, we show that 8TE inhibited TNF-alpha-induced activation of NF-kappa B and LPS-stimulated IL-6 in a dose- and time-dependent manner in Raw 264.7 macrophages. 8TE potently inhibited TNF-alpha-induced phosphorylation of transforming growth factor beta-activated kinase 1 (TAK1), an upstream kinase essential for the activation of NF-kappa B. Interestingly, 8TE significantly increased the expression of A20 and to a less extent, cylindromatosis (CYLD), both of which are inhibitors of NF-kappa B. The importance of induction of A20 in delta TE's anti-NF-kappa B effect is validated in A20 knockout cells where delta TE's inhibition of NF-kappa B was largely diminished. In pursuit of the cause for A20 induction, we found that delta TE treatment caused rapid and persistent elevation of dihydroceramides, while decreased ceramides initially but increased ceramides during prolonged treatment. These changes of sphingolipids were accompanied by increased cellular stress markers. Importantly, delta TE's induction of A20 and inhibition of NF-kappa B activation were partially counteracted by myriocin, a potent inhibitor of de novo synthesis of sphingolipids, indicating a critical role of sphingolipid modulation in delta TE-mediated effects. Since dihydroceramide has been shown to induce A20 and inhibit NF-kappa B in RAW cells, we conclude that that delta TE inhibits NF-kappa B activation by enhancing its negative regulator A20 as a result of modulating sphingolipids especially elevation of dihydroceramides. (C) 2018 Elsevier Inc. All rights reserved.