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

17β-Estradiol Prevents Early-Stage Atherosclerosis in Estrogen Receptor-Alpha Deficient Female Mice

Estrogen is atheroprotective and a high-affinity ligand for both known estrogen receptors, ERα and ERβ. However, the role of the ERα in early-stage atherosclerosis has not been directly investigated and is incompletely understood. ERα-deficient (ERα−/−) and wild-type (ERα+/+) female mice consuming an atherogenic diet were studied concurrent with estrogen replacement to distinguish the actions of 17β-estradiol (E2) from those of ERα on the development of early atherosclerotic lesions. Mice were ovariectomized and implanted with subcutaneous slow-release pellets designed to deliver 6 or 8 μg/day of exogenous 17β-estradiol (E2) for a period of up to 4 months. Ovariectomized mice (OVX) with placebo pellets (E2-deficient controls) were compared to mice with endogenous E2 (intact ovaries) and exogenous E2. Aortas were analyzed for lesion area, number, and distribution. Lipid and hormone levels were also determined. Compared to OVX, early lesion development was significantly (p < 0.001) attenuated by E2 with 55–64% reduction in lesion area by endogenous E2 and >90% reduction by exogenous E2. Compared to OVX, a decline in lesion number (2- to 4-fold) and lesser predilection (~4-fold) of lesion formation in the proximal aorta also occurred with E2. Lesion size, development, number, and distribution inversely correlated with circulating plasma E2 levels. However, atheroprotection was independent of ERα status, and E2 athero-protection in both genotypes was not explained by changes in plasma lipid levels (total cholesterol, triglyceride, and high-density lipoprotein cholesterol). The ERα is not essential for endogenous/exogenous E2-mediated protection against early-stage atherosclerosis. These observations have potentially significant implications for understanding the molecular and cellular mechanisms and timing of estrogen action in different estrogen receptor (ER) deletion murine models of atherosclerosis, as well as implications to human studies of ER polymorphisms and lipid metabolism. Our findings may contribute to future improved clinical decision-making concerning the use of hormone therapy

Loss of estrogen receptor β decreases mitochondrial energetic potential and increases thrombogenicity of platelets in aged female mice

Platelets derived from aged (reproductively senescent) female mice with genetic deletion of estrogen receptor beta (βER) are more thrombogenic than those from age-matched wild-type (WT) mice. Intracellular processes contributing to this increased thrombogenicity are not known. Experiments were designed to identify subcellular localization of estrogen receptors and evaluate both glycolytic and mitochondrial energetic processes which might affect platelet activation. Platelets and blood from aged (22–24 months) WT and estrogen receptor β knockout (βERKO) female mice were used in this study. Body, spleen weight, and serum concentrations of follicle-stimulating hormone and 17β-estradiol were comparable between WT and βERKO mice. Number of spontaneous deaths was greater in the βERKO colony (50% compared to 30% in WT) over the course of 24 months. In resting (nonactivated) platelets, estrogen receptors did not appear to colocalize with mitochondria by immunostaining. Lactate production and mitochondrial membrane potential of intact platelets were similar in both groups of mice. However, activities of NADH dehydrogenase, cytochrome bc1 complex, and cytochrome c oxidase of the electron transport chain were reduced in mitochondria isolated from platelets from βERKO compared to WT mice. There were a significantly higher number of phosphatidylserine-expressing platelet-derived microvesicles in the plasma and a greater thrombin-generating capacity in βERKO compared to WT mice. These results suggest that deficiencies in βER affect energy metabolism of platelets resulting in greater production of circulating thrombogenic microvesicles and could potentially explain increased predisposition to thromboembolism in some elderly females

A distal region of the human TGM1 promoter is required for expression in transgenic mice and cultured keratinocytes

BACKGROUND: TGM1(transglutaminase 1) is an enzyme that crosslinks the cornified envelope of mature keratinocytes. Appropriate expression of the TGM1 gene is crucial for proper keratinocyte function as inactivating mutations lead to the debilitating skin disease, lamellar ichthyosis. TGM1 is also expressed in squamous metaplasia, a consequence in some epithelia of vitamin A deficiency or toxic insult that can lead to neoplasia. An understanding of the regulation of this gene in normal and abnormal differentiation states may contribute to better disease diagnosis and treatment. METHODS: In vivo requirements for expression of the TGM1 gene were studied by fusing various lengths of promoter DNA to a reporter and injecting the DNA into mouse embryos to generate transgenic animals. Expression of the reporter was ascertained by Western blotting and immunohistochemistry. Further delineation of a transcriptionally important distal region was determined by transfections of progressively shortened or mutated promoter DNA into cultured keratinocytes. RESULTS: In vivo analysis of a reporter transgene driven by the TGM1 promoter revealed that 1.6 kilobases, but not 1.1 kilobases, of DNA was sufficient to confer tissue-specific and cell layer-specific expression. This same region was responsible for reporter expression in tissues undergoing squamous metaplasia as a response to vitamin A deprivation. Mutation of a distal promoter AP1 site or proximal promoter CRE site, both identified as important transcriptional elements in transfection assays, did not prevent appropriate expression. Further searching for transcriptional elements using electrophoretic mobility shift (EMSA) and transfection assays in cultured keratinocytes identified two Sp1 elements in a transcriptionally active region between -1.6 and -1.4 kilobases. While mutation of either Sp1 site or the AP1 site singly had only a small effect, mutation of all three sites eliminated nearly all the transcriptional activity. CONCLUSIONS: A distal region of the TGM1 gene promoter, containing AP1 and Sp1 binding sites, is evolutionarily conserved and responsible for high level expression in transgenic mice and in transfected keratinocyte cultures

DAX-1 expression in human breast cancer: comparison with estrogen receptors ER-α, ER-β and androgen receptor status

BACKGROUND: So far there have been no reports on the expression pattern of DAX-1 (dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1) in human breast cells and its relationship to the estrogen receptors, ER-α and ER-β, and the androgen receptor (AR). METHODS: In this study we evaluated, by immunohistochemistry and Western blot analysis, the presence and distribution of DAX-1 in benign breast disease (BBD), in situ carcinoma (CIS), and ductal and lobular breast carcinomas. RESULTS: In BBD and breast carcinomas, DAX-1 was present in both the nuclei and the cytoplasm of epithelial cells, although in infiltrative carcinomas the percentage of nuclear immunoreaction was higher than in CIS. An important relation was observed between DAX-1 and AR expression and between this orphan receptor and nodal status. CONCLUSION: DAX-1 might modify the AR and ER-β intracellular location, and because a direct positive relation between the expression of these three receptors was found it could be assumed that the presence of DAX-1 in neoplastic cells might indicate a possible failure of endocrine therapies

A Predominant Role for Parenchymal c-Jun Amino Terminal Kinase (JNK) in the Regulation of Systemic Insulin Sensitivity

It has been established that c-Jun N-terminal kinase 1 (JNK1) is essential to the pathogenesis of insulin resistance and type 2 diabetes. Although JNK influences inflammatory signaling pathways, it remains unclear whether its activity in macrophages contributes to adipose tissue inflammation and ultimately to the regulation of systemic metabolism. To address whether the action of this critical inflammatory kinase in bone marrow-derived elements regulates inflammatory responses in obesity and is sufficient and necessary for the deterioration of insulin sensitivity, we performed bone marrow transplantation studies with wild type and JNK1-deficient mice. These studies illustrated that JNK1-deficiency in the bone marrow-derived elements (BMDE) was insufficient to impact macrophage infiltration or insulin sensitivity despite modest changes in the inflammatory profile of adipose tissue. Only when the parenchymal elements lacked JNK1 could we demonstrate a significant increase in systemic insulin sensitivity. These data indicate that while the JNK1 activity in BMDE is involved in metabolic regulation and adipose milieu, it is epistatic to JNK1 activity in the parenchymal tissue for regulation of metabolic homeostasis

Estrogen-Dependent Gene Expression in the Mouse Ovary

Estrogen (E) plays a pivotal role in regulating the female reproductive system, particularly the ovary. However, the number and type of ovarian genes influenced by estrogen remain to be fully elucidated. In this study, we have utilized wild-type (WT) and aromatase knockout (ArKO; estrogen free) mouse ovaries as an in vivo model to profile estrogen dependent genes. RNA from each individual ovary (n = 3) was analyzed by a microarray-based screen using Illumina Sentrix Mouse WG-6 BeadChip (45,281 transcripts). Comparative analysis (GeneSpring) showed differential expression profiles of 450 genes influenced by E, with 291 genes up-regulated and 159 down-regulated by 2-fold or greater in the ArKO ovary compared to WT. Genes previously reported to be E regulated in ArKO ovaries were confirmed, in addition to novel genes not previously reported to be expressed or regulated by E in the ovary. Of genes involved in 5 diverse functional processes (hormonal processes, reproduction, sex differentiation and determination, apoptosis and cellular processes) 78 had estrogen-responsive elements (ERE). These analyses define the transcriptome regulated by E in the mouse ovary. Further analysis and investigation will increase our knowledge pertaining to how E influences follicular development and other ovarian functions

ESR1 and EGF genetic variation in relation to breast cancer risk and survival

The main purposes of this thesis were to analyse common genetic variation in candidate genes and candidate pathways in relation to breast cancer risk, prognosticators and survival, to develop statistical methods for genetic association analysis for evaluating the joint importance of genes, and to investigate the potential impact of adding genetic information to clinical risk factors for projecting individualised risk of developing breast cancer over specific time periods. In Paper I we studied genetic variation in the estrogen receptor α and epidermal growth factor genes in relation to breast cancer risk and survival. We located a region in the estrogen receptor α gene which showed a moderate signal for association with breast cancer risk but were unable to link common variation in the epidermal growth factor gene with breast cancer aetiology or prognosis. In Paper II we investigated whether suspected breast cancer risk SNPs within genes involved in androgen-to-estrogen conversion are associated with breast cancer prognosticators grade, lymph node status and tumour size. The strongest association was observed for a marker within the CYP19A1 gene with histological grade. We also found evidence that a second marker from the same gene is associated with histological grade and tumour size. In Paper III we developed a novel test of association which incorporates multivariate measures of categorical and continuous heterogeneity. In this work we described both a single-SNP and a global multi-SNP test and used simulated data to demonstrate the power of the tests when genetic effects differ across disease subtypes. In Paper IV we assessed the extent to which recently associated genetic risk variants improve breast cancer risk-assessment models. We investigated empirically the performance of eighteen breast cancer risk SNPs together with mammographic density and clinical risk factors in predicting absolute risk of breast cancer. We also examined the usefulness of various prediction models considered at a population level for a variety of individualised breast cancer screening approaches. The goal of a genetic association study is to establish statistical associations between genetic variants and disease states. Each variant linked to a disease can lead the way to a better understanding of the underlying biological mechanisms that govern the development of a disease. Increased knowledge of molecular variation provides the opportunity to stratify populations according to genetic makeup, which in turn has the potential to lead to improved disease prevention programs and improved patient care

Estrogenic Plant Extracts Reverse Weight Gain and Fat Accumulation without Causing Mammary Gland or Uterine Proliferation

Long-term estrogen deficiency increases the risk of obesity, diabetes and metabolic syndrome in postmenopausal women. Menopausal hormone therapy containing estrogens might prevent these conditions, but its prolonged use increases the risk of breast cancer, as wells as endometrial cancer if used without progestins. Animal studies indicate that beneficial effects of estrogens in adipose tissue and adverse effects on mammary gland and uterus are mediated by estrogen receptor alpha (ERα). One strategy to improve the safety of estrogens to prevent/treat obesity, diabetes and metabolic syndrome is to develop estrogens that act as agonists in adipose tissue, but not in mammary gland and uterus. We considered plant extracts, which have been the source of many pharmaceuticals, as a source of tissue selective estrogens. Extracts from two plants, Glycyrrhiza uralensis (RG) and Pueraria montana var. lobata (RP) bound to ERα, activated ERα responsive reporters, and reversed weight gain and fat accumulation comparable to estradiol in ovariectomized obese mice maintained on a high fat diet. Unlike estradiol, RG and RP did not induce proliferative effects on mammary gland and uterus. Gene expression profiling demonstrated that RG and RP induced estradiol-like regulation of genes in abdominal fat, but not in mammary gland and uterus. The compounds in extracts from RG and RP might constitute a new class of tissue selective estrogens to reverse weight gain, fat accumulation and metabolic syndrome in postmenopausal women