Estrogen receptor knockout mice as a model for endocrine research

The biological effects of estrogen in mammalian target tissues are important for multiple organ systems including the male and female reproductive tract and the neuroendocrine, skeletal, and cardiovascular systems. Numerous physiological effects of estradiol are modulated by the estrogen receptor (ER), a Class I member of the nuclear receptor superfamily. However, more recent studies have also implicated nongenomic effects of estrogen, which may involve a membrane-binding site. The two forms of the ER are the classical estrogen receptor-alpha (ERalpha) and the more recently discovered estrogen receptor-beta (ERbeta). Gene-targeting techniques were used to generate mice lacking either functional ERalpha (alphaERKO), ERbeta (betaERKO), or both ERs (alphabetaERKO) to provide a model for evaluating estrogen receptor action. These knockout models provide a unique tool to study the effects of estrogen in the context of the whole animal and to discern the role of each ER in various tissues. The reproductive phenotypes as well as some of the nonreproductive phenotypes of the different ERKO models are summarized.     

Interactions of nuclear estrogen receptor with DNA and RNA

The interaction of the nuclear estrogen receptor from hen oviduct with nucleic acids were studied by competition assay using DNa-cellulose centrifugation. We demonstrated that the estradiol-receptor complex binds similarly well to poly(A) RNA and denatured DNA. The estrogen receptor was found to interact more strongly with poly(G), poly(U) than with poly(A), poly(C). The receptor complex binds similarly to poly(A) and poly(dA), and to poly(U) and poly(dU). However, the receptor complex shows stronger binding to poly(G) than to poly(dG) and to poly(C) than to poly(dC). Studies with heteropolyribonucleotides indicated that poly(U1G1) is more effective in competing for the estrogen receptor, and poly(AC) and poly(AUG) are moderately effective, whereas poly(ACU) is least effective. GMP and dGMP showed some competition for the nuclear receptor at 300-fold higher nucleotide concentrations than that of the synthetic poly(G). Observations that the nuclear estrogen receptor binds to poly(A) RNA and interacts selectively with polyribonucleotides suggest that the estrogen receptor-RNA interaction may play a role for the function of estrogens in gene regulation.     

Cytosol and nuclear estrogen receptor binding in the preoptic area and hypothalamus of female rats during pregnancy and ovariectomized, nulliparous rats after steroid priming: correlation with maternal behavior

In a previous study, high nuclear estrogen receptor concentrations in the preoptic area (POA) were found on Day 16 of pregnancy to prime females to respond to a subsequent low dose of estradiol benzoate (EB) after hysterectomy-ovariectomy by exhibiting maternal behavior in 48 hr. Receptor concentrations in the POA were found to be higher than those in the hypothalamus (HYP). The present study investigated when nuclear estrogen receptors increase during pregnancy in POA and when the difference in receptor concentrations between POA and HYP occurs. An attempt was made to reproduce these pregnancy changes with a 16-day treatment of estrogen and progesterone in ovariectomized (OVX), nulliparous rats. In Experiment 1, we measured cytosol and nuclear estrogen receptor concentrations in the POA and HYP of female rats during pregnancy. Nuclear receptor concentrations in the POA increased beginning on Day 10, increased again on Day 16, and continued at this high level for the remainder of pregnancy. Nuclear estrogen receptor concentrations in the HYP remained at a lower level throughout most of pregnancy until Day 22 when they increased significantly. In Experiment 2, we tested the maternal behavior and measured estrogen receptor concentrations in OVX, steroid-primed, nulliparous rats after hysterectomy (H) and EB treatment. While 90% of estradiol (E) + progesterone (P)-primed females displayed short-latency maternal behavior 48 hr after H and EB treatment, 46% of E + vehicle (V)-treated controls were maternal. At 0 hr (prior to H and EB treatment), there was a significantly larger nuclear receptor accumulation in the POA but significantly attenuated receptor binding in the HYP. P treatment significantly affected cytosol and nuclear estrogen receptor dynamics. Differences in nuclear estrogen receptor concentrations were shown to be based on the number of available binding sites and not to changes in receptor affinity for estradiol.     

视黄醇结合蛋白RBP4可与多种核受体相互作用

视黄醇结合蛋白 (retinolbindingprotein ,RBP4 )是体内一种重要的转运蛋白 ,主要负责结合、转运全反式视黄醇 (维生素A ,VitA ) .VitA及其衍生物如11 cis 视黄醛、all trans 视黄酸等 ,均是体内非常重要的疏水分子 ,与视觉循环、胚胎发育等多种过程有关 .RBP4的功能障碍会导致     

Estrogens, phytoestrogens and colorectal neoproliferative lesions

Epidemiological and experimental studies suggest a protective role of estrogens against colorectal cancer. This effect seems to be mediated by their binding to estrogen receptor beta (ER-beta), one of the two estrogen receptors with high affinity for these hormones. Very recently, the demonstration of an involvement of ER-beta in the development of adenomatous polyps of the colon has also been documented, suggesting the use of selective ER-beta agonists in primary colorectal cancer prevention. Phytoestrogens are plant-derived compounds that structurally and functionally act as estrogen-agonists in mammals. They are characterized by a higher binding affinity to ER-beta as compared to estrogen receptor alpha (ER-alpha), the other estrogen receptor subtype. These biological characteristics explain why the administration of phytoestrogens does not produce the classical side effects associated to estrogen administration (cerebro- and cardio-vascular accidents, higher incidence of endometrial and breast cancer) and makes these substances ideal candidates for the prevention of colorectal cancer.     

Cytochemical detection of estrogen receptors in mammary tumours

The technical details of a cytochemical method for estrogen receptor identification in mammary tumours are presented and applied in 19 carcinomas and six adenofibromas. A fluorescent estradiol conjugate was used as receptor tracer. From the 19 mammary carcinomas, six were interpreted as estrogen receptor-negative (ER-), two were strongly receptor-positive (ER++) and eleven were moderately estrogen receptor-positive (ER+), having cells with a heterogeneous content of estrogen receptors. In fibroadenomas, the proliferating epithelia were moderately estrogen-receptor positive.     

In vitro mycotoxin binding to bovine uterine steroid hormone receptors

The mycotoxins, aflatoxin B(1), aflatoxin M(1), aflatoxicol and zearalenone were tested for binding to bovine endometrial estrogen and progestin receptors. Radioinert estradiol-17beta, estrone, testosterone, and cholesterol were evaluated for binding to the estrogen receptor. Zearalenone and aflatoxicol but not aflatoxins B(1) and M(1) competed with estradiol-17beta for the estrogen receptor. The order of binding affinities for the estrogen receptor were zearalenone > estradiol-17beta > estrone > aflatoxicol. The affinity of zearalenone for the estrogen receptor was 2-3 times that of estradiol-17beta. Progesterone, cortisol, radioinert R 5020, and cholesterol were evaluated for binding to the progestin receptor. None of the tested compounds except R 5020 and progesterone competed for the progestin receptor. The significance of aflatoxicol binding to the estrogen receptor is unclear. It is proposed that aflatoxicol binding to the receptor may alter gene expression in target tissues or act at the level of the hypothalamus to inhibit gonadotropin secretion and ovulation. These effects could explain reports of reduced fertility in domestic animals following ingestion of aflatoxin contaminated feedstuffs. It is also suggested that the mechanism of adverse effects on fertility of chronic aflatoxin ingestion in cattle and other livestock should be more thoroughly investigated.     

Regulatory effects of growth hormone, glucocorticoids, and thyroid hormone on the estrogen receptor level in the rat liver.

The multihormonal regulation of the estrogen receptor in the liver of female rats was studied under in vivo conditions. The steroid receptor level was assayed by hormone binding and specific mRNA analyzed by solution hybridization using a 35S-labeled RNA probe complementary to the ligand-binding domain of the estrogen receptor gene. Serum growth hormone levels were measured and correlated to the effects of glucocorticoid and thyroid hormone administration on the estrogen receptor expression. In animals subjected to adrenalectomy plus thyroidectomy, the estrogen receptor concentration was reduced from 59 fmol/mg cytosol protein to 10 fmol/mg protein (i.e., with 87% relative to control animals). Adrenalectomy or thyroidectomy alone caused a decrease with 14% and 66%, respectively. Substitution with 10 micrograms betamethasone and 1 microgram triiodothyronine daily for 9 days completely restored the receptor content to control levels. Substitution with either hormone alone increased, but only partially restored receptor levels. The effect of betamethasone alone was dose dependent from 10 micrograms/d to 100 micrograms/d. This dose dependence was not seen when the animal simultaneously received 1 microgram of triiodothyronine. Superphysiologic doses of triiodothyronine did not raise estrogen receptor levels above those seen in animals treated with physiologic doses. High doses of triiodothyronine (greater than 20 micrograms/d) decreased serum growth hormone levels. The estrogen receptor mRNA levels in livers from hypophysectomized animals were increased after treatment with growth hormone (2.5-fold), thyroid hormone (two-fold), and glucocorticoids (1.5-fold). The results obtained indicate a very complex regulation of liver estrogen receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Desmethylnafoxidine aziridine: an electrophilic affinity label for the estrogen receptor with high efficiency and selectivity

Desmethylnafoxidine aziridine (Naf-Az), an affinity label for the estrogen receptor based structurally on the antiestrogen nafoxidine, has been prepared in unlabeled and in high specific activity, tritium-labeled form and has been evaluated for its apparent competitive binding, and time-dependent irreversible, covalent attachment to the estrogen receptor. Naf-Az was synthesized through a key 1,2-diaryl-3,4-dihydronaphthalene intermediate that was prepared from 6-methoxy-1-tetralone by two routes involving alternate strategies for arylation. Conversion of the diaryldihydronaphthalene to Naf-Az through a series of deprotection-activation reactions culminated in ethyleneimine displacement of a methanesulfonate. The tritium-labeled material was prepared by tritium-iodine exchange on an iodinated methanesulfonate precursor, followed by ethyleneimine displacement. Compared to our previously-prepared reagent tamoxifen aziridine (Tam-Az), Naf-Az has a higher apparent competitive binding affinity, and it reacts with the estrogen receptor in cytosol preparations and in intact MCF-7 breast cancer cells rapidly and with at least comparable efficiency and selectivity. SDS-polyacrylamide gel electrophoretic analysis confirms its selective labeling of the Mr 66,000 estrogen receptor. Naf-Az should prove to be useful in studies aimed at characterizing the properties and structure of estrogen receptors.     

Lead identification of a potent benzopyranone selective estrogen receptor modulator

Starting from a phenol screening hit (6), three series of benzopyranone selective estrogen receptor modulators (SERMs) have been designed, synthesized, and analyzed for both estrogen receptor alpha binding affinity and in vitro activity in two cell assays. The lead compound identified, SP500263 (13), was more potent than raloxifene and tamoxifen in a cell-based assay measuring inhibition of interleukin-6 release.     

Heat shock 70 kDa protein 5/glucose-regulated protein 78 “AMP”ing up autophagy

Breast cancer is one of the most prevalent cancers in women, with more than 240,000 new cases reported in the United States in 2011. Classification of breast cancer based upon hormone and growth factor receptor profiling shows that approximately 70% of all breast cancers express estrogen receptor-α. Thus, drugs that either block estrogen biosynthesis (aromatase inhibitors like Letrozole), or compete with estrogen for estrogen receptor (ER) binding (selective ER modulators including tamoxifen; TAM) and/or cause ER degradation (selective estrogen receptor downregulators such as fulvestrant), are among the most prescribed targeted therapeutics for breast cancer. However, overall clinical benefit from the use of these drugs is often limited by resistance; ER⁺ breast cancers either fail to respond to endocrine therapies initially (de novo resistance), or they respond and then lose sensitivity over time (acquired resistance). While several preclinical studies postulate how antiestrogen resistance occurs, for the most part, the molecular mechanism(s) of resistance is unknown.     

Differential regulation of cyclic AMP synthesis by estrogen in MCF7 cells

The classical view of the molecular actions of estrogen is described by its interaction with the intracellular estrogen receptor (ER), the binding of hormone receptor complex to the estrogen response element (ERE) on the DNA and followed by the alterations of gene expressions. Recently it has been reported that membrane estrogen receptor (mER) exist and it is suggested to be G protein linked receptor. In this report we show that under steroid-free culture conditions supplemented with low percentage of charcoal-stripped serum, differential estrogen treatments of human breast cancer MCF7 cells induce different responses of cyclic AMP (cAMP) productions. Treating [2-(3)H]adenine-labeled MCF7 cells with 1 nM estrogen for 30 min stimulates cAMP production by measuring the ratio of [3H]cAMP:Total [3H]adenine nucleotides (ATP+ADP+cAMP), as determined by column chromatography, when compared with the control. This short-term estrogen treatment also significantly enhanced forskolin stimulated cAMP production when compared with the ratio of cAMP/Total measured in cells stimulated with forskolin alone. Pre-treating MCF7 cells with the same concentration of estrogen for 24h before the assay, on the contrary, significantly decreased the basal cAMP level and it also suppressed cAMP production stimulated with forskolin when compared with its respective value under short-term estrogen treatment. Estrogen receptor antagonist ICI 182780 abolished both the stimulatory and suppressive effect of estrogen on cAMP synthesis indicating both effects were mediated through ER. Pre-treating cells with pertussis toxin relieved the suppression of cAMP synthesis by chronic estrogen treatment. Our data suggest that estrogen exerts differential effects on the cAMP production in MCF7 cells, involving the activations Galpha(i) and Galpha(s) family of G proteins, depending on the length of time of hormone treatment.     

An immunocytochemical method for localization of estrogen receptors in rat tissues using a dinitrophenyl (DNP)-labeled rat monoclonal primary antibody.

We have developed an immunocytochemical method to demonstrate estrogen receptor in hormone-sensitive tissues of the rat using a dinitrophenyl (DNP) hapten-labeled rat antihuman estrogen receptor monoclonal antibody (MAb), H222. Mouse IgM anti-DNP was used secondarily, followed by a DNP/peroxidase conjugate, diaminobenzidine/hydrogen peroxide chromogen, and silver intensification. This method was applied to tissues from intact female rats and showed that estrogen receptor was localized in the nuclei of the stromal and glandular components of the uterine endometrium. Reduced receptor staining was observed in the luminal epithelium, with minimal myometrial staining. Anterior pituitary glands showed heterogeneous immunostaining and ovaries expressed the receptor predominantly in the interstitial cells; fallopian tubes demonstrated substantial epithelial staining. Uteri from chemically castrated rats showed reduced estrogen receptor immunostaining in both stromal and luminal cells, whereas staining was enhanced in the glandular elements. Classical estrogen-unresponsive tissues (heart, lung, and spleen) were unstained. Antibody controls involved pre-blocking antibody recognition sites on the receptor with unlabeled antibodies to estrogen receptor (H222, H226, and D547), as well as use of an inappropriate DNP-labeled antibody to metallothionein. These controls illustrated the specific nature of the DNP-H222 binding.     

Ultrastructural analysis of estrogen receptor immunoreactive neurons in the medial preoptic area of the female rat brain

Summary Neurons of the medial preoptic area were studied in the brain of the female rat by means of ultrastructural immunocytochemistry using a monoclonal antibody generated against purified estrogen receptor (ER), in order to delineate the morphological correlates of estrogen feedback mechanisms. In addition to the preoptic area, the bed nucleus of the stria terminalis, the arcuate and ventromedial nuclei of the hypothalamus exhibited an intense labelling for estrogen receptor. At the light microscopic level, the cell nuclei were immunoreactive. No major alterations were detected in the ER expression of medial preoptic neurons sampled during the estrous cycle, but proestrous rats did exhibit a slightly increased intensity of staining. At the ultrastructural level, the ER immunoreactivity was primarily confined to the nuclei and associated with the chromatin. Long term steroid deprivation elicited by either ovariectomy or ovariectomy plus adrenalectomy resulted in a marked intensity of nuclear labelling. This pattern was not influenced by acute estradiol replacement.These morphological data indicate that neurons of the medial preoptic area have the capacity to detect estrogens via receptor mechanisms and that changes in the level of the circulating ligand are manifested in an alteration in the staining for the estrogen receptor. The study also supports the revised concept of estrogen receptor action by demonstrating the presence of receptors in the nuclei of the cells, whether or not they are occupied by their ligand.Supported by grants from the IBRO/MacArthur Foundation Network Grant, the National Science Foundation (NSF INT 8703030), the Hungarian Academy of Sciences (OTKA 104), the National Institutes of Health (NS 19266), the National Foundation of Technical Development (OKKFT Tt 286/1986) and the Well-come Trust (14685/1.5)     

125I]iododesethyl tamoxifen aziridine: synthesis and covalent labeling of the estrogen receptor with an iodine-labeled affinity label

Iododesethyl tamoxifen aziridine (I-Tam-Az), an analog of the estrogen receptor-affinity label tamoxifen aziridine (Tam-Az) in which the ethyl group has been replaced by an iodine, has been prepared by two routes: (a) metallation of a bromotriarylethylene system, followed by reaction with iodine, and aziridinylation, and (b) direct iodination of a trimethylstannyl triarylethylene system that is the immediate precursor of I-Tam-Az. The latter method can be used to prepare [125I]I-Tam-Az rapidly and in good yield, both at carrier-added and no-carrier-added levels; specific activities greater than 200 Ci/mmol have been obtained. In competitive radiometric binding assays with the estrogen receptor, I-Tam-Az has an apparent affinity of ca. 20%, equivalent to that of Tam-Az. It also undergoes rapid and selective time-dependent, irreversible binding to the estrogen receptor. [125I]I-Tam-Az reacts covalently with estrogen receptor in uterine cytosol preparations; its attachment is rapid and efficient, but somewhat less selective than that of Tam-Az. Estrogen receptor in intact MCF-7 human breast cancer cells can also be labeled with [125I]I-Tam-Az, and autoradiographic analysis of salt extracts of labeled nuclear estrogen receptor on SDS-polyacrylamide slab gels shows highly selective labeling of a 65K protein. [125I]I-Tam-Az is an efficient, selective affinity label for the estrogen receptor, available at high specific activity, and should be useful in studies on estrogen receptor structure, dynamics, and chromatin interactions.