Estrogen receptor alpha (ERalpha) deficiency in macrophages results in increased stimulation of CD4+ T cells while 17beta-estradiol acts through ERalpha to increase IL-4 and GATA-3 expression in CD4+ T cells independent of antigen presentation

The effects of 17beta-estradiol (E2) on immune function have been extensively reported. The effects are dependent on concentration and duration of exposure and potential differences in signaling between the known E2 receptors, estrogen receptors (ER) alpha and ERbeta. Through the use of ER-deficient mice, we and others have begun to demonstrate the role of the two known receptors in modulating immune functional activities. Previous studies have shown that cells of the innate immune system have altered function (bactericidal capacity) and patterns of cytokine expression (increased proinflammatory cytokine expression) through amelioration of ERalpha signaling. In this study, we extend these studies to analysis of T cell differentiation and proliferation in APC-dependent and APC-independent in vitro assay systems. Our results demonstrate that ERalpha deficiency in splenic macrophages, but not CD11c+ splenic dendritic cells pulsed with OVA significantly enhances proliferative responses and IFN-gamma production by transgenic OVA peptide-specific (OT-II) CD4+ T cells when compared with Ag-pulsed APC from wild-type littermates. The addition of E2 in this culture system did not significantly affect the production of IFN-gamma. In addition, when purified CD4+ T cells from ERalpha-deficient and wild-type littermates were stimulated with anti-CD3/CD28 Ab in the absence of E2, there were no significant differences in IFN-gamma or IL-4 production. However, the addition of E2 significantly increased IL-4 secretion, as well as increased GATA-3 mRNA levels from ERalpha-replete CD4+ T cells, while this effect was abrogated in ERalpha-deficient CD4+ T cells.     

Estrogen receptor alpha/beta isoforms, but not betacx, modulate unique patterns of gene expression and cell proliferation in Hs578T cells

The actions of 17beta-estradiol (E2) and selective estrogen receptor modulators (SERMs) have been extensively investigated regarding their ability to act through estrogen receptor-alpha (ERalpha) to perturb estrogen receptor positive (ER+) breast cancer (BC) growth. However, many BCs also express ERbeta, along with multiple estrogen receptor (ER) splice variants such as ERbetacx, an ERbeta splice variant incapable of binding ligand. To gain a more comprehensive understanding of ER action in BC cells, we stably expressed ERalpha, ERbeta, or ERbetacx under doxycycline (Dox) control in Hs578T cells. Microarrays performed on E2 or 4OH-tamoxifen (4HT) treated Hs578T ERalpha and ERbeta cells revealed distinct ligand and receptor-dependent patterns of gene regulation, while the induction of ERbetacx did not alter gene expression patterns. E2 stimulation of Hs578T ERbeta cells resulted in a 27% decrease in cellular proliferation, however, no significant change in proliferation was observed following the exposure of Hs578T ERalpha or ERbeta cells to 4HT. Expression of ERbetacx in Hs578T cells did not effect cellular proliferation. Flow cytometry assays revealed a 50% decrease in E2-stimulated Hs578T ERbeta cells entering S-phase, along with a 17% increase in G0/G1 cell-cycle arrest. We demonstrate here that ERalpha and ERbeta regulate unique gene expression patterns in Hs578T cells, and such regulation likely is responsible for the observed isoform-specific changes in cell proliferation. Hs578T ER expressing cell-lines provide a unique BC model system, permitting the comparison of ERalpha, ERbeta, and ERbetacx actions in the same cell-line.     

Plasma membrane estrogen receptors exist and functions as dimers

A small pool of estrogen receptors (ERalpha and -beta) localize at the plasma membrane and rapidly signal to affect cellular physiology. Although nuclear ERs function mainly as homodimers, it is unknown whether membrane-localized ER exists or functions with similar requirements. We report that the endogenous ER isoforms at the plasma membrane of breast cancer or endothelial cells exist predominantly as homodimers in the presence of 17beta-estradiol (E2). Interestingly, in endothelial cells made from ERalpha /ERbeta homozygous double-knockout mice, membrane ERalpha or ERbeta are absent, indicating that the endogenous membrane receptors derive from the same gene(s) as the nuclear receptors. In ER-negative breast cancer cells or Chinese hamster ovary cells, we expressed and compared wild-type and dimer mutant mouse ERalpha. Only wild-type ERalpha supported the ability of E2 to rapidly activate ERK, cAMP, and phosphatidylinositol 3-kinase signaling. This resulted from E2 activating Gsalpha and Gqalpha at the membrane in cells expressing the wild-type, but not the dimer mutant, ERalpha. Intact, but not dimer mutant, ERalpha also supported E2-induced epidermal growth factor receptor transactivation and cell survival. We also confirmed the requirement of dimerization for membrane ER function using a second, less extensively mutated, human ERalpha. In summary, endogenous membrane ERs exist as dimers, a structural requirement that supports rapid signal transduction and affects cell physiology.     

Nature of functional estrogen receptors at the plasma membrane

Although rapid signaling by estrogen at the plasma membrane is established, it is controversial as to the nature of the receptor protein. Estrogen may bind membrane proteins comparable to classical nuclear estrogen receptors (ERs), but some studies identify nonclassical receptors, such as G protein-coupled receptor (GPR)30. We took several approaches to define membrane-localized estrogen-binding proteins. In endothelial cells (ECs) from ERalpha/ERbeta combined-deleted mice, estradiol (E2) failed to specifically bind, and did not activate cAMP, ERK, or phosphatidyinositol 3-kinase or stimulate DNA synthesis. This is in contrast to wild-type ECs, indicating the lack of any functional estrogen-binding proteins in ERalpha/ERbeta combined-deleted ECs. To directly determine the identity of membrane and nuclear-localized ER, we isolated subcellular receptor pools from MCF7 cells. Putative ER proteins were trypsin digested and subjected to tandem array mass spectrometry. The output analysis identified membrane and nuclear E2-binding proteins as classical human ERalpha. We also determined whether GPR30 plays any role in E2 rapid actions. MCF7 (ER and GPR30 positive) and SKBR-3 (ER negative, GPR30 positive) cells were incubated with E2. Only MCF7 responded with significantly increased signaling. In MCF7, the response to E2 was not different in cells transfected with small interfering RNA to green fluorescent protein or GPR30. In contrast, interfering RNA to ERalpha or ER inhibition prevented rapid signaling and resulting biology in MCF7. In breast cancer and ECs, nuclear and membrane ERs are the same proteins. Furthermore, classical ERs mediate rapid signals induced by E2 in these cells.     

Characterization of estrogen-responsive epithelial cell lines and their infectivity by genital Chlamydia trachomatis

Chlamydial attachment and infectivity in vitro and ascending disease and sequelae in vivo have been reported to be enhanced/modulated by estrogen. Endometrial carcinoma cell lines Ishikawa and HEC-1B and the breast cancer lines MCF-7 and HCC-1806 were examined for Chlamydia trachomatis E infectivity. Estrogen receptor (ER) presence was confirmed by Western blot and qRT-PCR analyses. FACS analysis was used to determine the percent of plasma membrane-localized ERs (mERs), and their activity was tested by estrogen binding and competitive estrogen antagonists assays. Chlamydiae grew in all cell lines with HEC (90%) > MCF-7 (57%)>Ishikawa (51%) > HCC-1806 (20%). The cell line ER isoform composition was re-defined as: ERalpha + ERbeta + for MCF-7, HCC-1806 and Ishikawa; and ERbeta only for HEC-1B. HeLa cells were also tested and found to express ERbeta, but not ERalpha. A small percentage of both ERs were surface-exposed and functionally active. The endometrium-predominant ERbeta isoform was found in all cell lines, including those most representative of the common sites of C. trachomatis infection. Thus, the role of chlamydial attachment/infectivity will now be analyzed in ERbeta+and-isogenic HEC-1B cells.     

A nongenomic action of 17beta-estradiol as the mechanism underlying the acute suppression of secretion of luteinizing hormone

The objective of the present study was to determine the ability of 17beta-estradiol (E(2)) and conjugated forms of E(2) (E(2) conjugated to BSA [E(2)-BSA] and a novel conjugate, E(2) conjugated to a small peptide [E(2)-PEP]) to prevent the GnRH-induced secretion of LH and to determine the role of estradiol receptors (ERs) and ER subtypes (ERalpha, also known as ESR1, and ERbeta, also known as ESR2) in the mediation of the acute action of E(2) in primary cultures of ovine pituitary cells. Preincubation of cells for 15 min with E(2), E(2)-BSA, or E(2)-PEP prevented the GnRH-induced secretion of LH (P < 0.01). Treatment of cells with nonestrogenic steroid hormones did not affect secretion of LH when given alone, nor did these steroids impair the E(2)-induced inhibition of LH secretion (P > 0.1). Likewise, treatment of cells with the ER-antagonists tamoxifen, hydroxytamoxifen, or ICI 182 780 did not affect (P > 0.1) secretion of LH when given alone but did prevent (P < 0.01) the inhibition by E(2) and the E(2)-conjugates on GnRH-induced secretion of LH. When cells were treated with subtype-selective ER agonists, the ERalpha agonist (propylpyrazole-triol), but not the ERbeta agonist (diarylpropionitrile), decreased (P < 0.01) the GnRH-induced secretion of LH. In conclusion, the rapidity by which E(2) prevented GnRH-induced release of LH in ovine pituitary cells suggests that this inhibition is mediated via a nongenomic action of E(2). The inhibition of GnRH-induced secretion of LH proved to be steroid specific and mediated by ERs. It may occur specifically through ERalpha. The fact that E(2)-BSA or E(2)-PEP mimicked the action of E(2) suggests that this effect was mediated by an ER associated with the plasma membrane.     

ERbeta shifts from mitochondria to nucleus during estrogen-induced neoplastic transformation of human breast epithelial cells and is involved in estrogen-induced synthesis of mitochondrial respiratory chain proteins

Both estrogen receptors (ER) alpha (ERalpha) and beta (ERbeta) are localized in the nucleus, plasma membrane, and mitochondria, where they mediate the different physiological effects of estrogens. It has been observed that the relative subcellular localization of ERs is altered in several cancer cells. We have demonstrated that MCF-10F cells, the immortal and non-tumorigenic human breast epithelial cells (HBEC) that are ERalpha-negative and ERbeta-positive, are transformed in vitro by 17beta-estradiol (E(2)), generating highly invasive cells that are tumorigenic in severe combined immunodeficient mice. E(2)-transformed MCF-10F (trMCF) cells exhibit progressive loss of ductulogenesis, invasive (bsMCF) and tumorigenic (caMCF) phenotypes. Immunolocalization of ERbeta by confocal fluorescent microscopy and electron microscopy revealed that ERbeta is predominantly localized in mitochondria of MCF-10F and trMCF cells. Silencing ERbeta expression with ERbeta-specific small interference RNA (siRNA-ERbeta) markedly diminishes both nuclear and mitochondrial ERbeta in MCF-10F cells. The ERbeta shifts from its predominant localization in the mitochondria of MCF-10F and trMCF cells to the nucleus of bsMCF cells, becoming predominantly nuclear in caMCF cells. Furthermore, we demonstrated that the mitochondrial ERbeta in MCF-10F cells is involved in E(2)-induced expression of mitochondrial DNA (mtDNA)-encoded respiratory chain (MRC) proteins. This is the first report of an association of changes in the subcellular localization of ERbeta with various stages of E(2)-induced transformation of HBEC and a functional role of mitochondrial ERbeta in mediating E(2)-induced MRC protein synthesis. Our findings provide a new insight into one of the potential roles of ERbeta in human breast cancer.     

Hepatic stellate cells contain the functional estrogen receptor beta but not the estrogen receptor alpha in male and female rats

In an earlier study, we showed that estradiol (E2) inhibits proliferation and transformation in cultured rat hepatic stellate cells (HSCs) and that the actions of E2 are mediated through estrogen receptors (ERs). This study reports on an investigation of the cellular localization of ER subtypes ERalpha and ERbeta using immunohistochemistry in experimental fibrotic liver rats and of each ER subtype expression in cultured rat HSCs by evaluating the produced mRNA and protein. The results indicate that high levels of ERbeta expression and low or no levels of ERalpha expression were observed in normal and fibrotic livers and in quiescent and activated HSCs from both males and females. The specificity of E2-mediated antiapoptotic induction through the ERbeta was shown by dose-dependent inhibition by the pure ER antagonist ICI 182,780 in HSCs which were undergoing early apoptosis. These findings demonstrate for the first time that rat HSCs possess functional Erbeta, but not Eralpha, to respond directly to E2 exposure.     

Differential effects of xenoestrogens on coactivator recruitment by estrogen receptor (ER) alpha and ERbeta

It has been proposed that tissue-specific estrogenic and/or antiestrogenic actions of certain xenoestrogens may be associated with alterations in the tertiary structure of estrogen receptor (ER) alpha and/or ERbeta following ligand binding; changes which are sensed by cellular factors (coactivators) required for normal gene expression. However, it is still unclear whether xenoestrogens affect the normal behavior of ERalpha and/or ERbeta subsequent to receptor binding. In view of the wide range of structural forms now recognized to mimic the actions of the natural estrogens, we have assessed the ability of ERalpha and ERbeta to recruit TIF2 and SRC-1a in the presence of 17beta-estradiol, genistein, diethylstilbestrol, 4-tert-octylphenol, 2',3',4', 5'-tetrachlorobiphenyl-ol, and bisphenol A. We show that ligand-dependent differences exist in the ability of ERalpha and ERbeta to bind coactivator proteins in vitro, despite the similarity in binding affinity of the various ligands for both ER subtypes. The enhanced ability of ERbeta (over ERalpha) to recruit coactivators in the presence of xenoestrogens was consistent with a greater ability of ERbeta to potentiate reporter gene activity in transiently transfected HeLa cells expressing SRC-1e and TIF2. We conclude that ligand-dependent differences in the ability of ERalpha and ERbeta to recruit coactivator proteins may contribute to the complex tissue-dependent agonistic/antagonistic responses observed with certain xenoestrogens.