Cross Talk in HEK293 Cells Between Nrf2, HIF, and NF-κB Activities upon Challenges with Redox Therapeutics Characterized with Single-Cell Resolution

Aim: Many transcription factors with importance in health and disease are redox regulated. However, how their activities may be intertwined in responses to redox-perturbing stimuli is poorly understood. To enable in-depth characterization of this aspect, we here developed a methodology for simultaneous determination of nuclear factor E2-related factor 2 (Nrf2), hypoxia-inducible factor (HIF), and nuclear factor kappa-light-chain-enhancer of activated B cell (NF-kappa B) activation at single-cell resolution, using a new tool named pTRAF (plasmid for transcription factor reporter activation based upon fluorescence). The pTRAF allowed determination of Nrf2, HIF, and NF-kappa B activities in a high-resolution and high-throughput manner, and we here assessed how redox therapeutics affected the activities of these transcription factors in human embryonic kidney cells (HEK293). Results: Cross talk was detected between the three signaling pathways upon some types of redox therapeutics, also by using inducers typically considered specific for Nrf2, such as sulforaphane or auranofin, hypoxia for HIF activation, or tumor necrosis factor alpha (TNF alpha) for NF-kappa B stimulation. Doxorubicin, at low nontoxic doses, potentiated TNF alpha-induced activation of NF-kappa B and HIF, without effects in stand-alone treatment. Stochastic activation patterns in cell cultures were also considerable upon challenges with several redox stimuli. Innovation: A novel strategy was here used to study simultaneous activation of Nrf2, HIF, and NF-kappa B in single cells. The method can also be adapted for studies of other transcription factors. Conclusion: The pTRAF provides new opportunities for in-depth studies of transcription factor activities. In this study, we found that upon challenges of cells with several redox-perturbing conditions, Nrf2, HIF, and NF-kappa B are uniquely responsive to separate stimuli, but can also display marked cross talk to each other within single cells.