Real‐time measurement of E2: ERα transcriptional activity in living cells

Kinetic analyses of diverse physiological processes have the potential to unveil new aspects of the molecular regulation of cell biology at temporal levels. 17beta-estradiol (E2) regulates diverse physiological effects by binding to the estrogen receptor alpha (ERalpha), which primarily works as a transcription factor. Although many molecular details of the modulation of ERalpha transcriptional activity have been discovered including the impact of receptor plasma membrane localization and its relative E2-evoked signaling, the knowledge of real-time ERalpha transcriptional dynamics in living cells is lacking. Here, we report the generation of MCF-7 and HeLa cells stably expressing a modified luciferase under the control of an E2-sensitive promoter, which activity can be continuously monitored in living cells and show that E2 induces a linear increase in ERalpha transcriptional activity. Ligand-independent (e.g., epidermal growth factor) receptor activation was also detected in a time-dependent manner. Kinetic profiles of ERalpha transcriptional activity measured in the presence of both receptor antagonists and inhibitors of ERalpha plasma membrane localization reveal a biphasic dynamic of receptor behavior underlying novel aspects of receptor-regulated transcriptional effects. Finally, analysis of the rate of the dose-dependent E2 induction of ERalpha transcriptional activity demonstrates that low doses of E2 induce an effect identical to that determined by high concentrations of E2 as a function of the duration of hormone administration. Overall, we present the characterization of sensitive stable cell lines were to study the kinetic of E2 transcriptional signaling and to identify new aspects of ERalpha function in different physiological or pathophysiological conditions.