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Nongenomic effects of estrogen receptor a (ER[alpha]) signaling have been described for decades. Several distinct animal models have been generated previously to analyze the nongenomic ER[alpha] signaling (eg, membrane-only ER, and ER[alpha]C451A). However, the mechanisms and physiological processes resulting solely from nongenomic signaling are still poorly understood. Herein, we describe a novel mouse model for analyzing nongenomic ER[alpha] actions named H2NES knock-in (KI). H2NES ER[alpha] possesses a nuclear export signal (NES) in the hinge region of ER[alpha] protein resulting in exclusive cytoplasmic localization that involves only the nongenomic action but not nuclear genomic actions. We generated H2NESKI mice by homologous recombination method and have characterized the phenotypes. H2NESKI homozygote mice possess almost identical phenotypes with ER[alpha] null mice except for the vascular activity on reendothelialization. We conclude that ER[alpha]-mediated nongenomic estrogenic signaling alone is insufficient to control most estrogen-mediated endocrine physiological responses; however, there could be some physiological responses that are nongenomic action dominant. H2NESKI mice have been deposited in the repository at Jax (stock no. 032176). These mice should be useful for analyzing nongenomic estrogenic responses and could expand analysis along with other ER[alpha] mutant mice lacking membrane-bound ER[alpha]. We expect the H2NESKI mouse model to aid our understanding of ER[alpha]-mediated nongenomic physiological responses and serve as an in vivo model for evaluating the nongenomic action of various estrogenic agents. Key Words: estrogen, estrogen receptor alpha, nongenomic action, extranuclear signaling, knock-in mutant mouse

Abstract Nongenomic effects of estrogen receptor α (ERα) signaling have been described for decades. Several distinct animal models have been generated previously to analyze the nongenomic ERα signaling (eg, membrane-only ER, and ERαC451A). However, the mechanisms and physiological processes resulting solely from nongenomic signaling are still poorly understood. Herein, we describe a novel mouse model for analyzing nongenomic ERα actions named H2NES knock-in (KI). H2NES ERα possesses a nuclear export signal (NES) in the hinge region of ERα protein resulting in exclusive cytoplasmic localization that involves only the nongenomic action but not nuclear genomic actions. We generated H2NESKI mice by homologous recombination method and have characterized the phenotypes. H2NESKI homozygote mice possess almost identical phenotypes with ERα null mice except for the vascular activity on reendothelialization. We conclude that ERα-mediated nongenomic estrogenic signaling alone is insufficient to control most estrogen-mediated endocrine physiological responses; however, there could be some physiological responses that are nongenomic action dominant. H2NESKI mice have been deposited in the repository at Jax (stock no. 032176). These mice should be useful for analyzing nongenomic estrogenic responses and could expand analysis along with other ERα mutant mice lacking membrane-bound ERα. We expect the H2NESKI mouse model to aid our understanding of ERα-mediated nongenomic physiological responses and serve as an in vivo model for evaluating the nongenomic action of various estrogenic agents.

Estrogen insensitivity syndrome (EIS) or estrogen resistance is very rare and most commonly caused by a mutation in ERα that results in the inability of estrogen to exert its biological effects. However, mutations in ESR1 gene and the underlying molecular mechanisms have not been thoroughly studied. We investigated the structural conformation, whole transcriptome, and DNA methylome for the ERα natural mutants, ESR1 Q375H and R394H that associated with EIS patients. We indicate that Q375H located in the coactivator binding pocket, while R394H located in the ligand binding pocket of the ERα ligand binding domain. Both mutants have changed the ERα structure conformation. We also demonstrate that both mutants differentially mediated whole transcriptome and DNA methylome aberrations in the genome. These mutants result in losing alteration to the estrogen-response genes predominantly when compared to WT ERα. To investigate the biological characterization of this natural mutation in vivo, a mouse model was generated harboring the human mutation, Esr1-Q379H (Esr1-Q), using CRISPR-/Cas9 and both male and female mice were analyzed. Our preliminary examinations show that the female and male Esr1-Q and αERKO mice were consistently more obese than the wild type (WT) mice. Female Esr1-Q mice have hemorrhagic cystic ovaries, rudimentary mammary ducts and hypoplastic uteri. In addition, the Esr1-Q mice have elevated levels of luteinizing hormone (LH) and nearly all examined phenotypes mirror those observed in αERKO mice. To test the responsiveness to estrogen in the Esr1-Q mice, a three-day bioassay with diethylstilbestrol (DES, a synthetic estrogen) was performed. WT uterine weight increased in the DES group when compared to controls. However, Esr1-Q uterine weight did not change with DES treatment, suggesting that this natural mutant lost ERα function. The female patient was also non-responsive to a high-dose estrogen treatment. The Esr1-Q male mice are infertile and show increased seminal vesicle weights as well as seminiferous tubule disruption in the testes. These findings provide an important basis for understanding the molecular and cellular mechanism of EIS and the mouse model offers a potential new way to study rare genetic receptor mutations in humans with hopes of developing a viable therapeutic approach.