An estrogen receptor-alpha knock-in mutation provides evidence of ligand-independent signaling and allows modulation of ligand-induced pathways in vivo

Estrogen-nonresponsive estrogen receptor-alpha (ER alpha) knock-in (ENERKI) mice were generated to distinguish between ligand-induced and ligand-independent ER-alpha actions in vivo. These mice have a mutation [glycine 525 to leucine (G525L)] in the ligand-binding domain of ER alpha, which significantly reduces ER alpha interaction with and response to endogenous estrogens, whereas not affecting growth factor activation of ligand-independent pathways. ENERKI mice had hypoplastic uterine tissues and rudimentary mammary gland ductal trees. Females were infertile due to anovulation, and their ovaries contained hemorrhagic cystic follicles because of chronically elevated levels of LH. The ENERKI phenotype confirmed that ligand-induced activation of ER alpha is crucial in the female reproductive tract and mammary gland development. Growth factor treatments induced uterine epithelial proliferation in ovariectomized ENERKI females, directly demonstrating that ER alpha ligand-independent pathways were active. In addition, the synthetic ER alpha selective agonist propyl pyrazole triol (PPT) and ERagonist diethylstilbestrol (DES) were still able to activate ligand-induced G525L ER alpha pathways in vitro. PPT treatments initiated at puberty stimulated ENERKI uterine development, whereas neonatal treatments were needed to restore mammary gland ductal elongation, indicating that neonatal ligand-induced ER alpha activation may prime mammary ducts to become more responsive to estrogens in adult tissues. This is a useful model for in vivo evaluation of ligand-induced ER alpha pathways and temporal patterns of response. DES did not stimulate an ENERKI uterotrophic response. Because ER beta may modulate ER alpha activation and have an antiproliferative function in the uterus, we hypothesize that ENERKI animals were particularly sensitive to DES-induced inhibition of ER alpha due to up-regulated uterine ER beta levels.