Sylvia C. Hewitt

Induction of Mammary Gland Development in Estrogen Receptor-α Knockout Mice

Mammary glands from the estrogen receptor-a knockout (alphaERKO) mouse do not undergo ductal morphogenesis or alveolar development. Disrupted ERalpha signaling may result in reduced estrogen-responsive gene products in the mammary gland or reduced mammotropic hormones that contribute to the alphaERKO mammary phenotype. We report that circulating PRL is reduced in the female alphaERKO mouse. Implantation of an age-matched, heterozygous ERalpha pituitary isograft under the renal capsule of 25-day-old or 12-week-old alphaERKO mice increased circulating PRL and progesterone levels, and induced mammary gland development. Grafted alphaERKO mice also possessed hypertrophied corpora lutea demonstrating that PRL is luteotropic in the alphaERKO ovary. By contrast, ovariectomy at the time of pituitary grafting prevented mammary gland development in alphaERKO mice despite elevated PRL levels. Hormone replacement using pellet implants demonstrated that pharmacological doses of estradiol induced limited mammary ductal elongation, and estradiol in combination with progesterone stimulated lobuloalveolar development. PRL alone or in combination with progesterone or estradiol did not induce alphaERKO mammary growth. Estradiol and progesterone are required for the structural development of the alphaERKO mammary gland, and PRL contributes to this development by inducing ovarian progesterone levels. Therefore, the manifestation of the alphaERKO mammary phenotype appears due to the lack of direct estrogen action at the mammary gland and an indirect contributory role of estrogen signaling at the hypothalamic/pituitary axis.

FOXA1 is an essential determinant of ERα expression and mammary ductal morphogenesis

FOXA1, estrogen receptor α (ERα) and GATA3 independently predict favorable outcome in breast cancer patients, and their expression correlates with a differentiated, luminal tumor subtype. As transcription factors, each functions in the morphogenesis of various organs, with ERα and GATA3 being established regulators of mammary gland development. Interdependency between these three factors in breast cancer and normal mammary development has been suggested, but the specific role for FOXA1 is not known. Herein, we report that Foxa1 deficiency causes a defect in hormone-induced mammary ductal invasion associated with a loss of terminal end bud formation and ERα expression. By contrast, Foxa1 null glands maintain GATA3 expression. Unlike ERα and GATA3 deficiency, Foxa1 null glands form milk-producing alveoli, indicating that the defect is restricted to expansion of the ductal epithelium, further emphasizing the novel role for FOXA1 in mammary morphogenesis. Using breast cancer cell lines, we also demonstrate that FOXA1 regulates ERα expression, but not GATA3. These data reveal that FOXA1 is necessary for hormonal responsiveness in the developing mammary gland and ERα-positive breast cancers, at least in part, through its control of ERα expression.

Uterine epithelial estrogen receptor α is dispensable for proliferation but essential for complete biological and biochemical responses.

Female fertility requires estrogen to specifically stimulate estrogen receptor α (ERα)-dependent growth of the uterine epithelium in adult mice, while immature females show proliferation in both stroma and epithelium. To address the relative roles of ERα in mediating estrogen action in uterine epithelium versus stroma, a uterine epithelial-specific ERα knockout (UtEpiαERKO) mouse line was generated by crossing Esr mice with Wnt7a-Cre mice. Expression of Wnt7a directed Cre activity generated selective deletion of ERα in uterine epithelium, and female UtEpiαERKO are infertile. Herein, we demonstrate that 17β-estradiol (E2)-induced uterine epithelial proliferation was independent of uterine epithelial ERα because DNA synthesis and up-regulation of mitogenic mediators were sustained in UtEpiαERKO uteri after E2 treatment. IGF-1 treatment resulted in ligand-independent ER activation in both wild-type (WT) and UtEpiαERKO and mimicked the E2 stimulatory effect on DNA synthesis in uterine epithelium. Uterine epithelial ERα was necessary to induce lactoferrin, an E2-regulated secretory protein selectively synthesized in the uterine epithelium. However, loss of uterine epithelial ERα did not alter the E2-dependent progesterone receptor (PR) down-regulation in epithelium. Strikingly, the uterine epithelium of UtEpiαERKO had robust evidence of apoptosis after 3 d of E2 treatment. Therefore, we surmise that estrogen induced uterine hyperplasia involves a dispensable role for uterine epithelial ERα in the proliferative response, but ERα is required subsequent to proliferation to prevent uterine epithelial apoptosis assuring the full uterine epithelial response, illustrating the differential cellular roles for ERα in uterine tissue and its contribution during pregnancy.

Activation of a Uterine Insulin-Like Growth Factor I Signaling Pathway by Clinical and Environmental Estrogens: Requirement of Estrogen Receptor-α

Recent data indicate that insulin-like growth factor I (IGF-I) may have a function in mediating the mitogenic effects of 17beta-estradiol (E2) in the uterus and in regulating the growth of uterine neoplasms. This study was designed to determine whether synthetic and plant-derived chemicals that interact with estrogen receptor-alpha (ERalpha) and elicit estrogenic responses also mimic E2 by activating the uterine IGF-I signaling pathway. Ovariectomized adult female mice were treated with both environmental and clinically relevant chemicals previously reported to display estrogenic and/or antiestrogenic properties, and their uteri were evaluated for an activated IGF-I signaling pathway. Diethylstilbestrol, 4-hydroxytamoxifen, the raloxifene analog LY353381, 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE), bisphenol A, and genistein were shown to mimic E2 in the uterus by increasing the level of IGF-I messenger RNA, inducing IGF-I receptor (IGF-IR) tyrosine phosphorylation, stimulating the formation of IGF-IR signaling complexes, and increasing both proliferating cell nuclear antigen expression and the number of mitotic cells in the epithelium. The dose of chemical necessary to activate IGF-I signaling varied, with the order of potency: E2 = diethylstilbestrol > LY353381 > 4-hydroxytamoxifen > genistein > HPTE > bisphenol A. Administration of the chemicals to ERalpha knockout mice did not activate IGF-IR, indicating that ERalpha is required for activation of uterine IGF-IR by these diverse chemicals. This study demonstrates that several chemicals shown previously to display estrogenic activities also mimic E2 by activating uterine IGF-I signaling.

Estrogen receptor transcription and transactivation: Estrogen receptor knockout mice - what their phenotypes reveal about mechanisms of estrogen action

Natural, synthetic and environmental estrogens have numerous effects on the development and physiology of mammals. Estrogen is primarily known for its role in the development and functioning of the female reproductive system. However, roles for estrogen in male fertility, bone, the circulatory system and immune system have been established by clinical observations regarding sex differences in pathologies, as well as observations following menopause or castration. The primary mechanism of estrogen action is via binding and modulation of activity of the estrogen receptors (ERs), which are ligand-dependent nuclear transcription factors. ERs are found in highest levels in female tissues critical to reproduction, including the ovaries, uterus, cervix, mammary glands and pituitary gland. Since other affected tissues have extremely low levels of ER, indirect effects of estrogen, for example induction of pituitary hormones that affect the bone, have been proposed. The development of transgenic mouse models that lack either estrogen or ER have proven to be valuable tools in defining the mechanisms by which estrogen exerts its effects in various systems. The aim of this article is to review the mouse models with disrupted estrogen signaling and describe the associated phenotypes.

Myeloid-specific estrogen receptor α deficiency impairs metabolic homeostasis and accelerates atherosclerotic lesion development

ERα is expressed in macrophages and other immune cells known to exert dramatic effects on glucose homeostasis. We investigated the impact of ERα expression on macrophage function to determine whether hematopoietic or myeloid-specific ERα deletion manifests obesity-induced insulin resistance in mice. Indeed, altered plasma adipokine and cytokine levels, glucose intolerance, insulin resistance, and increased adipose tissue mass were observed in animals harboring a hematopoietic or myeloid-specific deletion of ERα. A similar obese phenotype and increased atherosclerotic lesion area was displayed in LDL receptor-KO mice transplanted with ERα−/− bone marrow. In isolated macrophages, ERα was necessary for repression of inflammation, maintenance of oxidative metabolism, IL-4–mediated induction of alternative activation, full phagocytic capacity in response to LPS, and oxidized LDL-induced expression of ApoE and Abca1. Furthermore, we identified ERα as a direct regulator of macrophage transglutaminase 2 expression, a multifunctional atheroprotective enzyme. Our findings suggest that diminished ERα expression in hematopoietic/myeloid cells promotes aspects of the metabolic syndrome and accelerates atherosclerosis in female mice.

Estrogen-induced Proliferation of Uterine Epithelial Cells Is Independent of Estrogen Receptor α Binding to Classical Estrogen Response Elements

Acting via the estrogen receptor (ER), estradiol exerts pleomorphic effects on the uterus, producing cyclical waves of cellular proliferation and differentiation in preparation for embryo implantation. In the classical pathway, the ER binds directly to an estrogen response element to activate or repress gene expression. However, emerging evidence supports the existence of nonclassical pathways in which the activated ER alters gene expression through protein-protein tethering with transcription factors such as c-Fos/c-Jun B (AP-1) and Sp1. In this report, we examined the relative roles of classical and nonclassical ER signaling in vivo by comparing the estrogen-dependent uterine response in mice that express wild-type ERα, a mutant ERα (E207A/G208A) that selectively lacks ERE binding, or ERα null. In the compound heterozygote (AA/-) female, the nonclassical allele (AA) was insufficient to mediate an acute uterotrophic response to 17β-estradiol (E2). The uterine epithelial proliferative response to E2 and 4-hydroxytamoxifen was retained in the AA/-females, and uterine luminal epithelial height increased commensurate with the extent of ERα signaling. This proliferative response was confirmed by 5-bromo-2′-deoxyuridine incorporation. Microarray experiments identified cyclin-dependent kinase inhibitor 1A as a nonclassical pathwayresponsive gene, and transient expression experiments using the cyclin-dependent kinase inhibitor 1A promoter confirmed transcriptional responses to the ERα (E207A/G208A) mutant. These results indicate that nonclassical ERα signaling is sufficient to restore luminal epithelial proliferation but not other estrogen-responsive events, such as fluid accumulation and hyperemia. We conclude that nonclassical pathway signaling via ERα plays a critical physiologic role in the uterine response to estrogen.

Biological and biochemical consequences of global deletion of exon 3 from the ERα gene

To address issues resulting from α estrogen receptor-knockout (αERKO) residual N-terminal truncated estrogen receptor α, and to allow tissue-selective deletion of ERα, we generated loxP-flanked exon 3 mice. Initial characterization of global sox2 cre-derived exon 3-deleted Ex3αERKO mice indicated no ERα protein in uterine tissue and recapitulation of previously described female phenotypes, confirming successful ablation of ERα. Body weights of Ex3αERKO female mice were 1.4-fold higher than wild-tupe (WT) females and comparable to WT males. Microarray indicated the Ex3αERKO uterus is free of residual estrogen responses. RT-PCR showed Nr4a1 is increased 41-fold by estrogen in WT and 7.4-fold in αERKO, and not increased in Ex3αERKO. Nr4a1, Cdkn1a, and c-fos transcripts were evaluated in WT and Ex3αERKO mice following estrogen, IGF1, or EGF injections. All 3 were increased by all treatments in WT. None were increased by estrogen in Ex3αERKO. Nr4a1 increased 24.5- and 14.7-fold, Cdkn1a increased 14.2- and 12.3-fold, and c-fos increased 20.9-fold and 16.2-fold after IGF1 and EGF treatments, respectively, in the Ex3αERKO mice, confirming that growth factor regulation is independent of ERα. Our Ex3α ERα model will be useful in studies of complete or selective ablation of ERα in target tissues.

Extranuclear estrogen receptor-α stimulates NeuroD1 binding to the insulin promoter and favors insulin synthesis

Estrogen receptors (ERs) protect pancreatic islet survival in mice through rapid extranuclear actions. ERα also enhances insulin synthesis in cultured islets. Whether ERα stimulates insulin synthesis in vivo and, if so, through which mechanism(s) remain largely unknown. To address these issues, we generated a pancreas-specific ERα knockout mouse (PERαKO−/−) using the Cre-loxP strategy and used a combination of genetic and pharmacologic tools in cultured islets and β cells. Whereas 17β-estradiol (E2) treatment up-regulates pancreatic insulin gene and protein content in control ERαlox/lox mice, these E2 effects are abolished in PERαKO−/− mice. We find that E2-activated ERα increases insulin synthesis by enhancing glucose stimulation of the insulin promoter activity. Using a knock-in mouse with a mutated ERα eliminating binding to the estrogen response elements (EREs), we show that E2 stimulation of insulin synthesis is independent of the ERE. We find that the extranuclear ERα interacts with the tyrosine kinase Src, which activates extracellular signal-regulated kinases1/2, to increase nuclear localization and binding to the insulin promoter of the transcription factor NeuroD1. This study supports the importance of ERα in β cells as a regulator of insulin synthesis in vivo.

Estrogen-mediated Regulation of Igf1 Transcription and Uterine Growth Involves Direct Binding of Estrogen Receptor α to Estrogen-responsive Elements

Estrogen enables uterine proliferation, which depends on synthesis of the IGF1 growth factor. This proliferation and IGF1 synthesis requires the estrogen receptor (ER), which binds directly to target DNA sequences (estrogen-responsive elements or EREs), or interacts with other transcription factors, such as AP1, to impact transcription. We observe neither uterine growth nor an increase in Igf1 transcript in a mouse with a DNA-binding mutated ERα (KIKO), indicating that both Igf1 regulation and uterine proliferation require the DNA binding function of the ER. We identified several potential EREs in the Igf1 gene, and chromatin immunoprecipitation analysis revealed ERα binding to these EREs in wild type but not KIKO chromatin. STAT5 is also reported to regulate Igf1; uterine Stat5a transcript is increased by estradiol (E2), but not in KIKO or αERKO uteri, indicating ERα- and ERE-dependent regulation. ERα binds to a potential Stat5a ERE. We hypothesize that E2 increases Stat5a transcript through ERE binding; that ERα, either alone or together with STAT5, then acts to increase Igf1 transcription; and that the resulting lack of IGF1 impairs KIKO uterine growth. Treatment with exogenous IGF1, alone or in combination with E2, induces proliferation in wild type but not KIKO uteri, indicating that IGF1 replacement does not rescue the KIKO proliferative response. Together, these observations suggest in contrast to previous in vitro studies of IGF-1 regulation involving AP1 motifs that direct ERα-DNA interaction is required to increase Igf1 transcription. Additionally, full ERα function is needed to mediate other cellular signals of the growth factor for uterine growth.