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.
ABSTRACT
BACKGROUND: Endocrine-disrupting chemicals (EDCs) are suspected of altering estrogenic signaling through estrogen receptor (ER) α or ß (mERß1 in mice). Several EDC effects have been reported in animal studies and extrapolated to human studies. Unlike humans, rodents express a novel isoform of ERß (mERß2) with a modified ligand-binding domain sequence. EDC activity through this isoform remains uncharacterized. OBJECTIVES: We identified the expression pattern of mERß2 in mouse tissues and assessed the estrogenic activity of EDCs through mERß2. METHODS: mERß2 mRNA expression was measured in mouse tissues. HepG2 cells were used to assess the transactivation activity of mERß isoforms with EDCs and ER co-activators. 293A cells transiently transfected with mER isoforms were used to detect EDC-mediated changes in endogenous ER target gene expression. RESULTS: Expression of mERß2 mRNA was detected in mouse reproductive tissues (ovary, testis, and prostate) and lung and colon tissues from both female and male mice. Five (E2, DES, DPN, BPAF, Coum, 1-BP) of 16 compounds tested by reporter assay had estrogenic activity through mERß2. mERß2 had a compound-specific negative effect on ERß/ligand-mediated activity and ER target genes when co-expressed with mERß1. mERß2 recruited coactivators SRC2 or SRC3 in the presence of EDCs, but showed less recruitment than mERß1. CONCLUSION: mERß2 showed weaker estrogenic activity than mERß1 in our in vitro system, and can dampen mERß1 activity. In vivo models of EDC activity and ER-mediated toxicity should consider the role of mERß2, as rodent tissue responses involving mERß2 may not be reproduced in human biology.
