Development ofan Oxygen-Insensitive Nrf2 ReporterReveals Redox Regulation under Physiological Normoxia

Reactive oxygen species, particularly hydrogen peroxide (H₂O₂), play crucial roles in cellular signaling, with Nrf2 serving as a key transcription factor in maintaining redox homeostasis. However, the precise influence of H₂O₂ on Nrf2 activity under physiological normoxia remains unclear due to the limitations of oxygen-sensitive imaging methods. To address this, we developed and validated an oxygen-insensitive Nrf2 reporter named pericellular oxygen-insensitive Nrf2 transcriptional performance reporter (POINTER). We employed this reporter in human cerebral microvascular endothelial cells (hCMEC/D3). Using POINTER, we investigated how varying intracellular H₂O₂ concentrations affect Nrf2 regulation under normoxia (5 kPa O₂) compared to hyperoxia (ambient air, 21 kPa O₂). We manipulated intracellular H₂O₂ levels through exogenous application, chemogenetic production using a modified amino acid oxidase, and pharmacological induction with Auranofin. Our findings reveal that Nrf2 transcriptional activity is significantly lower under normoxia than under hyperoxia, supporting previous literature and expectations. Using POINTER, we found that both antioxidant pathway inhibition and sustained H₂O₂ elevation are essential for modulating Nrf2 activity. These findings provide new insights into the regulation of Nrf2 by H₂O₂.