PT  - JOURNAL ARTICLE
AU  - Marleen T. Aarts
AU  - Muriel Wagner
AU  - Tanne van der Wal
AU  - Antonius L. van Boxtel
AU  - Renée van Amerongen
TI  - A molecular toolbox to study progesterone receptor signaling
AID  - 10.1101/2023.07.20.549847
DP  - 2023 Jan 01
TA  - bioRxiv
PG  - 2023.07.20.549847
4099  - http://biorxiv.org/content/early/2023/07/20/2023.07.20.549847.short
4100  - http://biorxiv.org/content/early/2023/07/20/2023.07.20.549847.full
AB  - Progesterone receptor (PR) signaling is required for mammary gland development and homeostasis. A major bottleneck in studying PR signaling is the lack of sensitive assays to measure and visualize PR pathway activity both quantitatively and spatially. Here, we develop new tools to study PR signaling in human breast epithelial cells. First, we generate optimized Progesterone Responsive Element (PRE)-luciferase constructs and demonstrate that these new reporters are a powerful tool to quantify PR signaling activity across a wide range of progesterone concentrations in two luminal breast cancer cell lines, MCF7 and T47D. We also describe a fluorescent lentiviral PRE-GFP reporter as a novel tool to visualize PR signaling at the single-cell level. Our reporter constructs are sensitive to physiological levels of progesterone. Second, we show that low background signaling, and high levels of PR expression are a prerequisite for robustly measuring PR signaling. Increasing PR expression by transient transfection, stable overexpression in MCF7 or clonal selection in T47D, drastically improves both the dynamic range of luciferase reporter assays, and the induction of endogenous PR target genes as measured by qRT-PCR. We find that the PR signaling response differs per cell line, target gene and hormone concentration used. Taken together, our tools allow a more rationally designed approach for measuring PR signaling in breast epithelial cells.Competing Interest StatementThe authors have declared no competing interest.