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A molecular toolbox to study progesterone receptor signaling

View ORCID ProfileMarleen T. Aarts, View ORCID ProfileMuriel Wagner, View ORCID ProfileTanne van der Wal, View ORCID ProfileAntonius L. van Boxtel, View ORCID ProfileRenée van Amerongen
doi: https://doi.org/10.1101/2023.07.20.549847
Marleen T. Aarts
1Developmental, Stem Cell and Cancer Biology, Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, the Netherlands
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Muriel Wagner
1Developmental, Stem Cell and Cancer Biology, Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, the Netherlands
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Tanne van der Wal
1Developmental, Stem Cell and Cancer Biology, Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, the Netherlands
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Antonius L. van Boxtel
1Developmental, Stem Cell and Cancer Biology, Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, the Netherlands
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Renée van Amerongen
1Developmental, Stem Cell and Cancer Biology, Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, the Netherlands
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  • For correspondence: r.vanamerongen@uva.nl
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Abstract

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 Statement

The authors have declared no competing interest.

Footnotes

  • ↵# Twitter: @wntlab

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC 4.0 International license.
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Posted July 20, 2023.
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A molecular toolbox to study progesterone receptor signaling
Marleen T. Aarts, Muriel Wagner, Tanne van der Wal, Antonius L. van Boxtel, Renée van Amerongen
bioRxiv 2023.07.20.549847; doi: https://doi.org/10.1101/2023.07.20.549847
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A molecular toolbox to study progesterone receptor signaling
Marleen T. Aarts, Muriel Wagner, Tanne van der Wal, Antonius L. van Boxtel, Renée van Amerongen
bioRxiv 2023.07.20.549847; doi: https://doi.org/10.1101/2023.07.20.549847

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