Amentoflavone-induced oxidative stress activates NF-E2-related factor 2 via the p38 MAP kinase-AKT pathway in human keratinocytes

Abstract

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key transcription factor that responds to oxidative stress and xenobiotics. Multiple lines of evidence suggest that Nrf2 activation protects against aging, inflammation, and many diseases, including cancer. Nrf2 activators derived from natural sources have been widely studied. In this study, we investigated the effect of amentoflavone (AFN), a biflavonoid found in many plants, on Nrf2 signaling in human keratinocytes (HaCaT cells). AFN significantly increased ARE luciferase activity by Nrf2 accumulation in the nucleus. Subsequently, the levels of a Nrf2 target protein, NQO-1, were significantly increased by AFN in a dose- and time-dependent manner. To verify the mechanism of AFN-induced activation of Nrf2 signaling, we measured generation of reactive oxygen species (ROS). Interestingly AFN triggered mild ROS production. Additionally, AFN-induced Nrf2 activation was inhibited by N-acetyl cysteine. Therefore, we studied the effect of ROS-related signaling on Nrf2 by measuring the activation of AKT and members of the mitogen-activated protein kinase family, such as extracellular signal-regulated kinase (ERK1/2) and p38. The results showed that the pharmacological inhibitor of PI3K/AKT (LY294002) or p38 (SB 203580), but not ERK1/2 (U0126), abrogated AFN-induced Nrf2 activation. Subsequently, we found that silencing or inhibition of p38 resulted in decrease of AKT phosphorylation as well as inhibition of Nrf2 accumulation. Furthermore, we found that AFN stabilized Nrf2 by inhibiting its ubiquitination. Taken together, our results suggest that AFN contributes to Nrf2 activation through ROS-mediated activation of the p38-AKT pathway in HaCaT cells.