It is well recognized that our body has a comprehensive antioxidant system to counter oxidative stress. 4-HNE, an oxidative stressor, causes adaptive induction of detoxifying enzymes such as AKR1C1 and GSTA4 in different cell lines[18, 19], although the molecular pathway is not fully understood. The common feature of these detoxifying genes is that they have ARE-like sequences in their 5’-flanking regions[20, 21]. In this study, we demonstrate that NRF2 mediates 4-HNE induced gene expression of key antioxidant and detoxifying enzymes, resulting in enhanced 4-HNE metabolism.
4-HNE is a highly reactive electrophile, and several studies have reported that it is a potent NRF2 inducer[14, 15]. Our current results further confirmed that the nuclear translocation of NRF2 is significantly increased by 10 μM 4-HNE treatment in HeLa cells (Figure1). There are several mechanisms proposed for the 4-HNE-induced NRF2 activation. 4-HNE can react with cysteine sites in the KEAP1 protein and that may disrupt the KEAP1-dependent degradation of NRF2. In addition, 4-HNE may activate NRF2 through activation of upstream kinases such as protein kinase C, extracellular signal-regulated protein kinase and phosphoinositide 3-kinase[14, 23, 24]. In this study, we showed that 4-HNE could induce nuclear translocation of the NRF2 mutant lacking the KEAP1 binding domain (Figure1D), indicating that 4-HNE may have a direct effect on NRF2 itself. We found that 4-HNE could modify NRF2 protein in an in vitro testing system (Additional file1: Figure S1), and future studies will be needed to identify the specific amino acid sites modified by 4-HNE and their impacts in NRF2 signaling. In addition, several studies have reported that 4-HNE treatment leads to dramatic decrease of intracellular GSH[25, 26], and depletion of GSH can activate NRF2 signaling. In the present study, we showed that pretreatment of GSH could block the induction of ARE transcriptional activity by 4-HNE (Figure2B), suggesting that 4-HNE may activate NRF2 via depletion of GSH.
GSTA4 and AKR1C1 are two important enzymes for detoxification of 4-HNE. In the large GST family, GSTA4 is the most active isoform in catalyzing conjugation of GSH to 4-HNE. The higher expression level of GSTA4 in DU145 prostate cancer cells is associated with faster 4-HNE metabolism rate, compared to PC3 or LNCaP prostate cancer cells. It is also reported that overexpression of GSTA4 protects HepG2 cells from 4-HNE mediated oxidative injuries. AKR1C1 has high catalytic activity in reducing 4-HNE to less toxic 1,4-dihydroxynonenol. The role of HO-1 in the detoxification of 4-HNE is not clear. The induction of HO-1 may enhance the overall cellular antioxidant capacity and prevent oxidative stress induced cytotoxicity. Therefore, the induction of gene expression of these cellular protective enzymes by 4-HNE appears to be an adaptive response to enhance elimination of 4-HNE and reduce its toxicity. The transcriptional induction of these detoxifying and antioxidant genes is attenuated when NRF2 is knocked down (Figure3C), indicating that the induction is mediated by NRF2.