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

Abstract

Kinetic analyses of diverse physiological processes have the potential to unveil new aspects of the molecular regulation of cell biology at temporal levels. 17β-estradiol (E2) regulates diverse physiological effects by binding to the estrogen receptor α (ERα), which primarily works as a transcription factor. Although many molecular details of the modulation of ERα transcriptional activity have been discovered including the impact of receptor plasma membrane localization and its relative E2-evoked signalling, the knowledge of real-time ERα 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 ERα transcriptional activity. Ligand-independent (e.g., epidermal growth factor) receptor activation was also detected in a time-dependent manner. Kinetic profiles of ERα transcriptional activity measured in the presence of both receptor antagonists and inhibitors of ERα plasma membrane localization reveals a biphasic dynamic of receptor behaviour underlying novel aspects of receptor-regulated transcriptional effects. Finally, analysis of the rate of the dose-dependent E2 induction of ERα 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 where to study the kinetic of E2 transcriptional signaling and to identify new aspects of ERα function in different physiological or pathophysiological conditions.

A functional genetic screen for metabolic proteins unveils GART and the de novo purine biosynthetic pathway as novel targets for the treatment of luminal A ERα expressing primary and metastatic invasive ductal carcinoma

“…The ERα is a transcription factor, which regulates in an E2-dependent manner the expression of those genes that contain or not the estrogen response element (ERE) in their promoter region ( 40 ). In turn, we evaluated the impact of GART inhibition on the E2-dependent control of the expression of some ERE-containing (i.e., presenilin2 – pS2 and cathepsin D – Cat D) ( 16 ) and some non-ERE containing (i.e., cyclin D1 – CycD1 and Bcl-2) ( 44 ) genes in MCF-7 cells treated either with GART siRNA oligonucleotides or with LMX. The complete anti-estrogen fulvestrant (i.e., ICI182,780 - ICI) was also introduced as an internal control.…”

“…Notably, both ICI and LMX reduced the pS2 and CatD protein abundance in Y537S cells ( Figure 6C ; Supplementary Figures 4A, B ). Finally, we tested the ERα activity on a synthetic ERE-containing reporter gene stably transfected in MCF-7 cells ( 16 ) in the presence and the absence of both LMX, ICI, and E2. As expected ( 40 ), E2 increased the ERα transcriptional activity, which was completely prevented both in the presence and in the absence of ICI ( Figure 6D ).…”

“…MCF-7 and Y537S cells were stably transfected with a plasmid containing an ERE-nanoluciferase (NLuc)-PEST reporter gene and measurement of NLuc-PEST expression ( i.e. , ERα transcriptional activity) was performed after 24 hours of compound administration as described ( 16 , 26 ).…”

“…For growth curves, the xCELLigence DP system ACEA Biosciences, Inc. (San Diego, CA) Multi-E-Plate station was used to measure the time-dependent response to the indicated drugs by real-time cell analysis (RTCA), as previously reported ( 13 , 16 , 24 , 28 ). Synergy studies and Tam growth curves were done using Crystal Violet staining as reported in ( 29 ).…”