An oestrogen membrane receptor participates in estradiol actions for the prevention of amyloid-beta peptide1-40-induced toxicity in septal-derived cholinergic SN56 cells

Although oestrogen [17 beta-estradiol (E2)]-related neuroprotection has been demonstrated in different models, the involvement of non-classical oestrogen receptors (ERs) remains unexplored. Using the SN56 cholinergic cell line, we present evidence indicating that an ER associated with the plasma membrane participates in oestrogen-dependent inhibition of cell death induced by amyloid-beta peptide (A beta) toxicity. Similarly to E2 alone, a 15-min exposure to estradiol-horseradish peroxidase (E-HRP) significantly reduced A beta-induced cell death. This effect was decreased by the ER antagonist ICI 182,780 as well as by MC-20 antibody directed to a region neighbouring the ligand-binding domain of ER alpha. Using confocal microscopy on unpermeabilized SN56 cells exposed to MC-20 antibody, we identified a protein at the plasma membrane level. Western blot analysis of purified SN56 cell membrane fractions using MC-20 antibody revealed the presence of one band with the same electrophoretic mobility as intracellular ER alpha. Using conjugated forms of the steroid, E-HRP and E2 conjugated to bovine serum albumin-FITC, we demonstrated by confocal microscopy that SN56 cells contain surface binding sites for E2. Binding of both conjugates was blocked by pre-incubation with E2 and decreased by either ICI 182,780 or MC-20 antibody in a concentration-dependent manner. Thus, a membrane-related ER that shares some structural homologies with ER alpha may participate in oestrogen-mediated neuroprotection.     

Survival versus apoptotic 17beta-estradiol effect: role of ER alpha and ER beta activated non-genomic signaling

The capability of 17beta-estradiol (E2) to induce the non-genomic activities of its receptors (ER alpha and ER beta) and to evoke different signaling pathways committed to the regulation of cell proliferation has been analyzed in different cell cancer lines containing transfected (HeLa) or endogenous (HepG2, DLD1) ER alpha or ER beta. In these cell lines, E2 induced different effects on cell growth/apoptosis in dependence of ER isoforms present. The E2-ER alpha complex rapidly activated multiple signal transduction pathways (i.e., ERK/MAPK, PI3K/AKT) committed to both cell cycle progression and apoptotic cascade prevention. On the other hand, the E2-ER beta complex induced the rapid and persistent phosphorylation of p38/MAPK which, in turn, was involved in caspase-3 activation and cleavage of poly(ADP-ribose)polymerase, driving cells into the apoptotic cycle. In addition, the E2-ER beta complex did not activate any of the E2-ER alpha-activated signal molecules involved in cell growth. Taken together, these results demonstrate the ability of ER beta isoform to activate specific signal transduction pathways starting from plasma membrane that may justify the effect of E2 in inducing cell proliferation or apoptosis in cancer cells. In particular this hormone promotes cell survival through ER alpha non-genomic signaling and cell death through ER beta non-genomic signaling.     

Effect of estrogens on the interferon-gamma producing cell population of mouse splenocytes

We examined the effect of 17beta-estradiol (E2) on the cytokine production by mouse splenocytes. The production of interferon-gamma (IFN-gamma) was enhanced by E2 stimulation. E2 increased the number of cells expressing IFN-gamma and the IFN-gamma mRNA expression level in the cells. There were low- and high-level cells expressing IFN-gamma in the population. The natural killer (NK) cells and NKT cells were the low-level cells expressing IFN-gamma, and the number of these cells was increased by E2 stimulation. In addition, it was suggested that the enhancing effect of IFN-gamma production by E2 was mediated through estrogen receptor (ER) alpha, and ERbeta agonist stimulated IFN-gamma production. ERs are expressed on plasma membrane as well as in nucleus. The ligand specific to plasma membrane-associated ER (mER) enhanced the IFN-gamma production. In conclusion, our results indicated that E2 up-regulated the IFN-gamma production by mediating ERalpha, ERbeta and mERs.     

Estradiol receptors in breast cancer cells: Associated co-factors as targets for new therapeutic approaches

Estrogen receptors α (ERα) and β (ERβ) are nuclear receptors which transduce estradiol (E2) response in many tissues including the mammary gland and breast cancers (BC). They activate or inhibit specific genes involved in cell cycle progression and cell survival through multiple enzyme activities leading to malignant transformation. Hormone therapy (antiestrogens (AEs) and aromatase inhibitors (AIs) have been widely used to block the mitogenic action of E2 in patients with ER-positive BC. ERs act in concert with numerous other proteins outside and inside the nucleus where co-activators such as histone modifying enzymes help reaching optimum gene activation. Moreover, E2-mediated gene regulation can occur through ERs located at the plasma membrane or G protein-coupled estrogen receptor (GPER), triggering protein kinase signaling cascades. Classical AEs as well as AIs are inefficient to block the cascades of events emanating from the membrane and from E2 binding to GPER, leading patients to escape anti-hormone treatments and hormone therapy resistance. Many pathways are involved in resistance, mostly resulting from over-expression of growth factor membrane receptors, in particular the HER2/ErbB2 which can be inhibited by specific antibodies or tyrosine kinases inhibitors. Together with the Hsp90 molecular chaperone machinery, a complex interplay between ERs, co-activators, co-repressors and growth factor-activated membrane pathways represents potent targets which warrant to be manipulated alone and in combination to designing novel therapies. The discovery of new potential targets arising from micro array studies gives the opportunity to activate or inhibit different new ER-modulating effectors for innovative therapeutic interventions.     

Monoclonal antibodies to distinctive epitopes on the alpha and beta subunits of the fibronectin receptor

Monoclonal antibodies (MAbs) have been developed that can recognize epitopes that are unique to either the alpha or beta subunit of the fibronectin receptor (FnR). MAbs 11B4 and 7A8 immunoblot the alpha subunit of FnR either in purified form from Chinese hamster ovary (CHO) cells or in nonionic detergent extracts of cells of human and rodent origin electrophoresed under reducing or nonreducing conditions. The MAbs seem to be more reactive to the subunit when it has been electrophoresed under reducing conditions, suggesting that the epitope may be partially masked by the conformation conferred by disulfide bonding. A second set of MAbs, 7E2 and 7F9, is directed to an epitope on the beta subunit that is conformationally dependent upon disulfide bonding, as reduction of the subunit leads to loss of reactivity with both MAbs. Further, 7E2/7F9 immunoblots of nonionic detergent extracts of CHO cells, run under nonreducing conditions, reveal the presence of a third band (90-kDa), immunologically related to the beta subunit, which is not surface-labeled with 125I in intact cells and which does not copurify with the alpha and beta subunits isolated by immunoaffinity purification of FnR using the MAb PB1. The 90-kDa component is not found associated with a plasma membrane fraction prepared by crude cell fractionation, but is abundant in a low-speed pellet containing nuclei and intracellular membranes. This finding suggests that the 90-kDa component is a precursor to the beta subunit. Finally, the epitope of 7E2/7F9 is unique to CHO cells, as cross-reactivity to other cell types cannot be demonstrated by either immunoblotting or immunoprecipitation.     

Estrogen receptors in the central nervous system and their implication for dopamine-dependent cognition in females

This article is part of a Special Issue “Estradiol and cognition”.Over the past 30 years, research has demonstrated that estrogens not only are important for female reproduction, but also play a role in a diverse array of cognitive functions. Originally, estrogens were thought to have only one receptor, localized exclusively to the cytoplasm and nucleus of cells. However, it is now known that there are at least three estrogen receptors (ERs): ERα, ERβ and G-protein coupled ER1 (GPER1). In addition to being localized to nuclei, ERα and ERβ are localized to the cell membrane, and GPER1 is also observed at the cell membrane. The mechanism through which ERs are associated with the membrane remains unclear, but palmitoylation of receptors and associations between ERs and caveolin are implicated in membrane association. ERα and ERβ are mostly observed in the nucleus using light microscopy unless they are particularly abundant. However, electron microscopy has revealed that ERs are also found at the membrane in complimentary distributions in multiple brain regions, many of which are innervated by dopamine inputs and were previously thought to contain few ERs. In particular, membrane-associated ERs are observed in the prefrontal cortex, dorsal striatum, nucleus accumbens, and hippocampus, all of which are involved in learning and memory. These findings provide a mechanism for the rapid effects of estrogens in these regions. The effects of estrogens on dopamine-dependent cognition likely result from binding at both nuclear and membrane-associated ERs, so elucidating the localization of membrane-associated ERs helps provide a more complete understanding of the cognitive effects of these hormones.     

Expression and localization of estrogen receptor alpha in the C2C12 murine skeletal muscle cell line

The classical model of 17beta-estradiol action has been traditionally described to be mediated by the estrogen receptor (ER) localized exclusively in the nucleus. However, there is increasing functional evidence for extra nuclear localization of ER. We present biochemical, immunological and molecular data supporting mitochondrial-microsomal localization of ER alpha in the C2C12 skeletal muscle cell line. We first established [(3)H]17beta estradiol binding characteristics in whole cells in culture. Specific and saturable [(3)H]17beta estradiol binding sites of high affinity were then detected in mitochondrial fractions (K(d) = 0.43 nM; B(max) = 572 fmol/mg protein). Immunocytological studies revealed that estrogen receptors mainly localize at the mitochondrial and perinuclear level. These results were also confirmed using fluorescent 17beta estradiol-BSA conjugates. The immunoreactivity did not translocate into the nucleus by 17beta-estradiol treatment. Western and Ligand blot approaches corroborated the non-classical localization. Expression and subcellular distribution of ER alpha proteins were confirmed in C2C12 cells transfected with ER alpha siRNA and by RT-PCR employing specific primers. The non-classical distribution of native pools of ER alpha in skeletal muscle cells suggests an alternative mode of ER localization/function.     

Evidence of an estrogen receptor form devoid of estrogen binding ability in MCF-7 cells

In MCF-7 breast cancer cells, hydroxytamoxifen (OH-Tam) up-regulates the estrogen receptor (ER) in a form unable to bind [(3)H]estradiol (E(2)). We show here that this property is not restricted to this antiestrogen. [(3)H]E(2) binding assays (whole cell assays, DCC assays on cell extracts) and enzyme immunoassays (Abbott) performed in parallel, establish the permanent presence of such unusual ERs in the absence of any exposure of the cells to a ligand. E(2) and the pure antiestrogen RU 58 668, which down-regulate ER, also decrease [(3)H]E(2) binding. In control cells, these ERs represent about the half of the whole receptor population; they also display a tendency to stabilize within the cell nucleus. Loss of E(2) binding ability appears irreversible, since we failed to label receptor accumulated under OH-Tam with [(3)H]E(2) or [(3)H]tamoxifen aziridine (TAZ). Cycloheximide (CHX), which blocks E(2)-induced down regulation of ER, failed to stabilize [(3)H]E(2) binding (whole cell assay) after an [(3)H]E(2) pulse (1 h), confirming that regulation of E(2) binding and peptide level are related to different regulatory mechanisms. Loss of binding ability could not be ascribed to any ER cleavage as demonstrated by Western blotting with a panel of ER antibodies raised against its various domains (67 kDa ER solely detected). We propose that loss of E(2) binding ability is related to the aging process of the receptor, i.e. it is progressively converted to a form devoted to degradation after it has accomplished its physiological role. Ligands may favor (E(2), RU 58 668) or impede (OH-Tam) this elimination process.     

Clonal analysis of B and T cell responses to Ia antigens. IV. Proliferative T cell clones recognizing E beta and/or E alpha allodeterminants

The allospecific T cell recognition of the I-Ek molecule was assessed by using eight A. TH anti-A. TL proliferative T cell clones, all of which expressed the Thy-1-2+, Lyt-1+, Lyt-2-, Ia-, and p94,180+ cell surface phenotype. The use of panels of stimulating cells from homozygous of F1 hybrid strains indicated each T cell clone exhibited specificity for distinct alloactivating determinants including: i) a private E beta k-controlled determinant expressed in cis- or trans-complementing E beta kE alpha strains; ii) an apparently nonpolymorphic E alpha determinant resembling the serologic specificity Ia.7, i.e., present in all strains carrying E alpha and E beta expressor alleles; and iii) a series of conformational I-E determinants, the expression of which required a precisely defined combinatorial association of E beta plus E alpha chains. Two clones were found to be reactivated by cis- but not trans-complementing E beta k E alpha k strains, and another recognized an allodeterminant shared by the I-Ab molecule. Various I-Ek-reactive monoclonal antibodies (mAb) directed to epitopes presumably expressed on either E alpha (epitope clusters I and II) or E beta (epitope cluster III) chains inhibited the proliferative responses of seven clones recognizing private E beta k or unique E beta E alpha conformational activating determinants. By contrast, the restimulation of the clone directed to a nonpolymorphic E alpha determinant was selectively blocked by anti-Ia.7 mAb defining epitopes on the E alpha chains but not by those directed to the E beta chain. On the basis of these data, it was concluded that the recognition sites of most anti-I-Ek proliferative T cells were expressed on the E beta chain or the E beta plus E alpha interaction products, and that a minority of such alloreactive T cells could be activated through recognition of the E alpha chain per se.     

Estrogen regulation of human osteoblast function is determined by the stage of differentiation and the estrogen receptor isoform

Although osteoblasts have been shown to respond to estrogens and express both isoforms of the estrogen receptor (ER alpha and ER beta), the role each isoform plays in osteoblast cell function and differentiation is unknown. The two ER isoforms are known to differentially regulate estrogen-inducible promoter-reporter gene constructs, but their individual effects on endogenous gene expression in osteoblasts have not been reported. We compared the effects of 17 beta-estradiol (E) and tamoxifen (TAM) on gene expression and matrix formation during the differentiation of human osteoblast cell lines stably expressing either ER alpha (hFOB/ER alpha 9) or ER beta (hFOB/ER beta 6). Expression of the appropriate ER isoform in these cells was confirmed by northern and western blotting and the responses to E in the hFOB/ER beta 6 line were abolished by an ER beta-specific inhibitor. The data demonstrate that (1) in both the hFOB/ER cell lines, certain responses to E or TAM (including alkaline phosphatase, IL-6 and IL-11 production) are more pronounced at the late mineralization stage of differentiation compared to earlier stages, (2) E exerted a greater regulation of bone nodule formation and matrix protein/cytokine production in the ER alpha cells than in ER beta cells, and (3) the regulated expression of select genes differed between the ER alpha and ER beta cells. TAM had no effect on nodule formation in either cell line and was a less potent regulator of gene/protein expression than E. Thus, both the ER isoform and the stage of differentiation appear to influence the response of osteoblast cells to E and TAM.     

Expression of oestrogen receptor-beta in invasive breast tumours

Steroid hormone receptors are used routinely to predict endocrine responsiveness in patients with breast cancer. Two oestrogen receptors (ERs): ER alpha and ER beta have been identified. Although ER alpha and ER beta genes share a large degree of homology, it is generally thought that their distribution and function are substantially different in many tissues. Both of them may be expressed in normal and neoplastic tissues of the breast. While much is known about ER alpha, the role of ER beta is still undefined, especially at the protein level. Recent development of reliable antibodies to ER beta has provided opportunity to test immunohistochemical reactions detecting ER beta in archival breast tumours. The aim of our study was to learn more about the cellular mechanisms underlying the relationship of ER beta and progesterone receptor (PR) in breast cancer tissues, discriminating between hormone-dependent and hormone-independent tumours. ER alpha and PR content of tumour tissues of 154 patients with breast cancer were tested by in situ indirect immunohistochemical method parallel with ligand binding biochemical assay. ER beta was detected in 8 ER alpha-/PR+ breast carcinomas by immunohistochemical method too. Steroid hormone receptor content was analysed comparing to the histologic type and grading of the tumours. CONCLUSIONS: A considerable part of breast carcinomas belongs to the ER+/PR+ and ER-/PR- groups. About 1-2% of the tumours is expected to be ER alpha-/ER beta+/PR+ type. In such cases ER alpha negative reaction together with PR positivity can signal the necessity of the immunohistochemical detection of ER beta in routine histopathological practice, presenting the precise steroid hormone receptor status for the most effective endocrine therapy of the patients.