Nrf2 degron-fused reporter system: a new tool for specific evaluation of Nrf2 inducers

The transcription factor Nrf2 is degraded through the proteasome pathway, but is stabilized in response to oxidative and electrophilic stresses, and activates cytoprotective enzyme genes through binding to the antioxidant/electrophile response element (ARE/EpRE). Nrf2 inducers thus have considerable potential as therapeutic drugs. Although ARE-driven reporters are commonly employed to validate Nrf2 inducers, these reporters are relatively nonspecific. We have generated a new reporter, Nrf2d-LacZ, which may prove to be a better tool for validation of Nrf2 inducers. We made the Nrf2d-LacZ reporter by fusing the N-terminus of Nrf2 harboring a Neh2 degron to beta-galactosidase (LacZ), and compared its activity in immortalized mouse embryo fibroblasts (MEFs) with conventional ARE-luciferase (ARE-Luc) reporters in 293T cells, and in MEFs. Nrf2d-LacZ was degraded in unstressed conditions, but stabilized upon exposure to stresses. LacZ activity was induced by electrophiles in a dose-dependent manner, and the induction was detected much more rapidly compared with ARE-Luc. Nrf2d-LacZ was activated not only by electrophiles but also by a variety of other Nrf2 inducing stresses. Although ARE-Luc was activated by 12-O-Tetradecanoylphorbol 13-acetate in an Nrf2-independent manner, Nrf2d-LacZ was not activated by TPA, thus emphasizing the specificity of the Nrf2d-LacZ reporter system for validation of Nrf2 inducers.