Abstract
Estrogen receptor-α (ERα) plays an important role in normal and abnormal physiology of the human reproductive system by interacting with the endogenous ligand estradiol (E2). However, other ligands, either analogous or dissimilar to E2, also bind to ERα. This may create unintentional activation of ER signaling in reproductive tissues that can lead to cancer development. We developed a transgenic mouse model that constitutively expresses a firefly luciferase (FLuc) split reporter complementation biosensor (NFLuc-ER-LBDG521T-CFLuc) to simultaneously evaluate the dynamics and potency of ligands that bind to ERα. We first validated this model using various ER ligands, including Raloxifene, Diethylstilbestrol, E2, and 4-hydroxytamoxifen, by employing FLuc-based optical bioluminescence imaging of living mice. We then used the model to investigate the carcinogenic property of Bisphenol A (BPA), an environmental estrogen, by long-term exposure at full and half environmental doses. We showed significant carcinogenic effects on female animals while revealing activated downstream ER signaling as measured by bioluminescence imaging. BPA induced tumor-like outgrowths in female transgenic mice, histopathologically confirmed to be neoplastic and epithelial in origin. This transgenic mouse model expressing an ERα folding-biosensor is useful in evaluation of estrogenic ligands and their downstream effects, and in studying environmental estrogen induced carcinogenesis in vivo.
Highlights
Estrogen receptors (ERs: ERαand ERβ) are vital hormone receptors mediating the regulation of cell growth and differentiation in response to estrogens
After 24 h the cells were treated with various ligands for 16 h (Fig. 1b) and subjected to a luminometer assay for renilla luciferase (RLuc) and firefly luciferase (FLuc) activity using coelenterazine and LARII substrates, respectively
Since we used an E2 non-responsive ER-mutant (ER G521T) for vector construction, ligands such as DES, OHT, methyl-piperidinopyrazole (MPP) and Ral induced significantly high intramolecular folding and subsequent FLuc complementation signals, while various E2 analogs (E1, E2, and E3) showed very low levels of FLuc complementation (Fig. 1b). These results clearly demonstrated that the ligand-induced ER-intramolecular folding biosensor expressed under the control of a CAG promoter is specific and sensitive for imaging conformational changes of the ER ligand-binding domains (LBDs) in response to different ligands in living cells (Fig. 1b)
Summary
Estrogen receptors (ERs: ERαand ERβ) are vital hormone receptors mediating the regulation of cell growth and differentiation in response to estrogens. ERαand ERβare the two receptors identified to date that dispatch estrogen-mediated signaling in mammalian cells These two ERs have nearly identical amino acid sequences, approximately 97% similarity in DNA-binding domains, 56% in ligand-binding domains (LBDs), and 24% in their N-terminal ends. To study the ligand-induced downstream effects of ERs26 and the in vivo dynamics of ERα27, transgenic mice expressing luciferase under the control of the estrogen response element (ERE) have been developed previously. At this time there are no transgenic mouse models developed so far to screen ligands that alter the function of ER and simultaneously show the ligand binding affinity as well as its potency
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