17-Oxo-DHA Induces Heme Oxygenase-1 Expression in Mouse Epidermal JB6 Cells through Nrf2 Activation

Abstract

Recently, growing attention has been given to the discovery of new classes of bioactive mediators derived from omega-3 polyunsaturated fatty acids (PUFAs), such as docosahexaenoic acid (DHA) and to their role as endogenous anti-inflammatory signaling mediators. 17-Oxo-DHA is an electrophilic metabolite of DHA, generated by the action of cyclooxygenase-2 (COX-2) and a dehydrogenase. Owing to its electrophilic nature, 17-oxo-DHA modulates multiple redox-sensitive signaling mechanisms including Nrf2-induced anti-oxidant response. In the present study, the molecular mechanism responsible for activation of Nrf2-Keap1 signaling in response to 17-oxo-DHA treatment was investigated in mouse epidermal JB6 cells. 17-Oxo-DHA significantly enhanced nuclear translocation and transcriptional activity of Nrf2 and its target protein heme oxygenase-1 (HO-1). Notably, 17-oxo-DHA was a more potent Nrf2 activator than its parent molecule, DHA. Knockdown of Nrf2 in JB6 cells diminished the 17-oxo-DHA induced upregulation of HO-1. Similar results were obtained with experiments using embryofibroblasts from Nrf2 knockout mice, corroborating that Nrf2 is essential for 17-oxo-DHA induced upregulation of HO-1 expression. 17-Oxo-DHA also markedly reduced the expression of Keap1 protein in posttranslational levels by inducing ubiquitination of Keap1. In identifying a critical Keap1 cysteine residue as a potential target of 17-oxo-DHA, I showed that mutation of Cys151 in Keap1 not only abolished the 17-oxo-DHA induced expression of HO-1, but also 17-oxo-DHA-induced ubiquitination of Keap1, suggesting that 17-oxo-DHA may interact with this cysteine residue directly. Interestingly, Keap1 degradation in response to 17-oxo-DHA coincided with p62 accumulation. 17-oxo-DHA-induced Keap1 degradation was abolished by an autophagy inhibitor, suggesting that Keap1 degradation is associated with p62-mediated autophagy. Treatment with 17-oxo-DHA generated high molecular weight (HMW) form of Keap1 in HEK293 cells harboring wild-type FLAG-Keap1, whereas in cells harboring C151S mutant form of Keap1, HMW forms were not observed. Taken together, Cys151 residue of Keap1 appears to be a critical target of 17-oxo-DHA in its induction of antioxidant expression and Keap1 degradation. Our results provide insight into 17-oxo-DHA as a novel and potent endogenous bioactive lipid mediator involved in anti-oxidant response.