Summary Estrogens regulate body weight and reproduction primarily through actions on estrogen receptor-α (ERα). However, ERα-expressing cells mediating these effects are not identified. We demonstrate that brain-specific deletion of ERα in female mice causes abdominal obesity stemming from both hyperphagia and hypometabolism. Hypometabolism and abdominal obesity, but not hyperphagia, are recapitulated in female mice lacking ERα in hypothalamic steroidogenic factor-1 (SF1) neurons. In contrast, deletion of ERα in hypothalamic pro-opiomelanocortin (POMC) neurons leads to hyperphagia, without directly influencing energy expenditure or fat distribution. Further, simultaneous deletion of ERα from both SF1 and POMC neurons causes hypometabolism, hyperphagia and increased visceral adiposity. Additionally, female mice lacking ERα in SF1 neurons develop anovulation and infertility, while POMC-specific deletion of ERα inhibits negative feedback regulation of estrogens and impairs fertility in females. These results indicate that estrogens act on distinct hypothalamic ERα neurons to regulate different aspects of energy homeostasis and reproduction.

Our data demonstrate that estrogens, estrogen receptor-α (ERα), and estrogen receptor-β (ERβ) regulate adipose tissue distribution, inflammation, fibrosis, and glucose homeostasis, by determining that αERKO mice have increased adipose tissue inflammation and fibrosis prior to obesity onset. Selective deletion of adipose tissue ERα in adult mice using a novel viral vector technology recapitulated the findings in the total body ERα null mice. Generation of a novel mouse model, lacking ERα specifically from adipocytes (AdipoERα), demonstrated increased markers of fibrosis and inflammation, especially in the males. Additionally, we found that the beneficial effects of estrogens on adipose tissue require adipocyte ERα. Lastly, we determined the role of ERβ in regulating inflammation and fibrosis, by breeding the AdipoERα into the βERKO background and found that in the absence of adipocyte ERα, ERβ has a protective role. These data suggest that adipose tissue and adipocyte ERα protects against adiposity, inflammation, and fibrosis in both males and females.

The estrogen receptor-␣ (ER␣) is a critical transcription factor that regulates epithelial cell proliferation and ductal morphogenesis during postnatal mammary gland development. Tissue recombination and transplantation studies using the first generation of ER␣ knockout (ERKO) mice suggested that this steroid hormone receptor is required in the mammary stroma that subsequently exerts its effect on the epithelium through additional paracrine signaling events. A more detailed analysis revealed that ERKO mice produce a truncated ER␣ protein with detectable transactivation activity, and it is likely that this functional ER␣ variant has masked the biological significance of this steroid receptor in the mammary epithelium. In this article, we describe the generation a Cre-loxbased conditional knockout of the ER␣ gene to study the biological function of this steroid receptor in the epithelial compartment at defined stages of mammary gland development. The mouse mammary tumor virus (MMTV)-Cre-mediated, epithelial-specific ablation of exon 3 of the ER␣ gene in virgin mice severely impaired ductal elongation and side branching. The conditional knockout resulted in ablation of the ER␣ protein, and the progesterone receptor (PR), whose expression is under the control of ER␣, was largely absent. The whey acidic protein (WAP)-Cre-mediated deletion of ER␣ during successive gestation cycles resulted in a loss of ductal side-branching and lobuloalveolar structures, ductal dilation, and decreased proliferation of alveolar progenitors. These abnormalities compromised milk production and led to malnourishment of the offspring by the second lactation. These observations suggest that ER␣ expression in the mammary epithelium is essential for normal ductal morphogenesis during puberty and alveologenesis during pregnancy and lactation.conditional knockout ͉ mammary gland

Reduced bioavailability of estrogen increases skeletal fracture risk in postmenopausal women, but the mechanisms by which estrogen regulates bone mass are incompletely understood. Because estrogen signaling in bone acts, in part, through estrogen receptor alpha (ERa), mice with global deletion of ERa (ERaKO) have been used to determine the role of estrogen signaling in bone biology. These animals, however, have confounding systemic effects arising from other organs, such as increased estrogen and decreased insulin-like growth factor 1 (IGF-1) serum levels, which may independently affect bone. Mice with tissue-specific ERa deletion in chondrocytes, osteoblasts, osteocytes, or osteoclasts lack the systemic effects seen in the global knockout, but show that presence of the receptor is important for the function of each cell type. Although bone mass is reduced when ERa is deleted from osteoblasts, no study has determined if this approach reduces whole bone strength. To address this issue, we generated female osteoblast-specific ERaKO mice (pOC-ERaKO) by crossing mice expressing a floxed ERa gene (ERa fl/fl ) with mice transgenic for the osteocalcin-Cre promoter (OC-Cre). Having confirmed that serum levels of estrogen and IGF-1 were unaltered, we focused on relating bone mechanics to skeletal phenotype using whole bone mechanical testing, microcomputed tomography, histology, and dynamic histomorphometry. At 12 and 18 weeks of age, pOC-ERaKO mice had decreased cancellous bone mass in the proximal tibia, vertebra, and distal femur, and decreased cortical bone mass in the tibial midshaft, distal femoral cortex, and L5 vertebral cortex. Osteoblast activity was reduced in cancellous bone of the proximal tibia, but osteoclast number was unaffected. Both femora and vertebrae had decreased whole bone strength in mechanical tests to failure, indicating that ERa in osteoblasts is required for appropriate bone mass and strength accrual in female mice. This pOC-ERaKO mouse is an important animal model that could enhance our understanding of estrogen signaling in bone cells in vivo.

Estrogen receptor α (ERα) plays an important role in the onset and progression of breast cancer, whereas p53 functions as a major tumor suppressor. We previously reported that ERα binds to p53, resulting in inhibition of transcriptional regulation by p53. Here, we report on the molecular mechanisms by which ERα suppresses p53's transactivation function. Sequential ChIP assays demonstrated that ERα represses p53-mediated transcriptional activation in human breast cancer cells by recruiting nuclear receptor corepressors (NCoR and SMRT) and histone deacetylase 1 (HDAC1). RNAi-mediated down-regulation of NCoR resulted in increased endogenous expression of the cyclin-dependent kinase (CDK)-inhibitor p21 Waf1/Cip1 (CDKN1A) gene, a prototypic transcriptional target of p53. While 17β-estradiol (E2) enhanced ERα binding to p53 and inhibited p21 transcription, antiestrogens decreased ERα recruitment and induced transcription. The effects of estrogen and antiestrogens on p21 transcription were diametrically opposite to their known effects on the conventional ERE-containing ERα target gene, pS2/TFF1. These results suggest that ERα uses dual strategies to promote abnormal cellular proliferation: enhancing the transcription of ERE-containing proproliferative genes and repressing the transcription of p53-responsive antiproliferative genes. Importantly, ERα binds to p53 and inhibits transcriptional activation by p53 in stem/progenitor cell-containing murine mammospheres, suggesting a potential role for the ER-p53 interaction in mammary tissue homeostasis and cancer formation. Furthermore, retrospective studies analyzing response to tamoxifen therapy in a subset of patients with ER-positive breast cancer expressing either wild-type or mutant p53 suggest that the presence of wild-type p53 is an important determinant of positive therapeutic response.nuclear receptor corepressor | mammary epithelial cells | mammospheres | tumor suppressor protein | tamoxifen therapy