Insulated Pathway Reporter Transposon Allows for HTS Approach to Transcriptional Activation Dynamics in Mammalian Cells

Cells respond to external stimuli through transient activation of multiple transcription factors (TF) downstream of signaling pathway networks. It has long been recognized that not only the nature of a stimulus, such as a stressor, but also its magnitude and duration define behavior and a fate of the responding organism. Surprisingly, there have been virtually no studies of cellular stress‐induced multiple TF activation in a detailed time‐dose‐response layout. Such studies would require a practical approach allowing for rapid generation of stable TF activity reporters in any cell type and performing the experiment in a high‐throughput screening (HTS) format. In order to expediently generate a large pool of functional pathway reporters based on established cell lines, we employed a reporter assembly containing a TF response element in the promoter for luciferase gene, followed by a promoter for continuous co‐expression of GFP/selecting antibiotic resistance, a pair of flanking insulators, and a pair of terminal piggyBac transposon sequences (1). A practical HTS workflow has been developed, which allows for about 2600 biological samples simultaneously processed in 96‐well plates per one worker and which uses only minimal equipment. We tested the approach for profiling time‐dose‐response of TF activation in lung (A549), kidney (NRK‐52E), and monocyte/macrophage (THP‐1) cell cultures treated with bacterial toxin pyocyanin (PCN) and a detoxicant eugenol. Of twenty stress‐related pathway reporters tested, the strongest responses to PCN were displayed by the oxidant/electrophile response pathway (TF: Nrf2), heat shock (TF: HSF1), and unfolded protein (TF: ATF6) reporters, indicating that cellular mechanisms of protection from PCN cytotoxicity were related primarily to cytosol and endoplasmic reticulum. Accordingly, pyocyanin, at doses corresponding to the TF activation maxima, induced massive formation of non‐lysosomal vacuoles in the cells, with a marginal degree of apoptosis. On the other hand, maximal activation of a p53 reporter was relatively weak, early, and located at subtoxic PCN concentrations, suggesting participation of p53 in the cell fate decision rather than chromatin protection. There was a significant activation of metal response (TF: MTF‐1) and hypoxia‐inducible (TF: HIF‐1α) reporters at elevated PCN concentrations, implying the iron‐mobilizing potential of the toxin, as well as a substantial aromatic hydrocarbon response (TF: AhR) at subtoxic PCN doses. A comparative analysis of the time‐dose‐response patterns for the tested pathway reporters in cells treated with PCN, hydrogen peroxide and eugenol suggested non‐ROS‐mediated cytotoxicity and oxidative deactivation of the toxin. In conclusion, a practical systematic approach to the regulatory signaling studies at the TF activity level has been demonstrated. Support or Funding Information Funding through the University of Missouri, Experiment Station Chemical Laboratories.