Nitric oxide activates Nrf2 through S-nitrosylation of Keap1 in PC12 cells

Nitric oxide (NO) exerts bifunctional effects on cell survival. While a high concentration of NO is cytotoxic, a relatively low concentration of NO promotes cytoprotection and cell survival. However, the molecular mechanism underlying the cytoprotective effect of NO remains poorly understood. One of the transcription factors that confer cellular protection against oxidative stress is NF-E2-related factor 2 (Nrf2), which is sequestered in the cytoplasm by forming an inactive complex with Klech-like ECH-associated protein 1 (Keap1). Previous studies suggested that various stimuli could induce the dissociation of Nrf2 from Keap1 in cytosol and/or promote its nuclear translocation by activating several upstream kinases. NO-mediated thiol modification in Keap1 has also been proposed as a possible mechanism of Nrf2 activation. Since NO can modify the function or activity of target proteins through S-nitrosylation of cysteine, we attempted to investigate whether the cytoprotective effect of NO is mediated through Nrf2 activation by directly modifying cysteine residues of Keap1. Our present study reveals that treatment of rat pheochromocytoma (PC12) cells with an NO donor S-nitroso-N-acetylpenicillamine (SNAP) induced nuclear translocation and DNA binding of Nrf2. Under the same experimental conditions, there was NO-mediated S-nitrosylation of Keap1 observed, which coincided with the Nrf2 activation. Moreover, SNAP treatment caused phosphorylation of Nrf2, and pharmacological inhibition of protein kinase C (PKC) abolished the phosphorylation and nuclear localization of Nrf2. In conclusion, NO can activate Nrf2 by S-nitrosylation of Keap1 and alternatively by PKC-catalyzed phosphorylation of Nrf2 in PC12 cells.

► Nitric oxide (NO) at physiologic concentrations exerts cytoprotective effects.
► Nrf2 is a master redox switch that upregulates many cytoprotective gene expression.
► In this study we found that NO induces S-nitrosylation of Keap1, a repressor of Nrf2.
► This facilitates dissociation of Nrf2 from Keap1 and nuclear translocation.
► In conclusion, NO can activate Nrf2 through S-nitrosylation of Keap1 in PC12 cells.