Araceli Morales

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

Although oestrogen [17β‐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‐β peptide (Aβ) toxicity. Similarly to E2 alone, a 15‐min exposure to estradiol‐horseradish peroxidase (E‐HRP) significantly reduced Aβ‐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 ERα. 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 ERα. 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 ERα may participate in oestrogen‐mediated neuroprotection.

Estradiol modulates acetylcholine-induced Ca2+ signals in LHRH-releasing GT1-7 cells through a membrane binding site.

Estrogen regulation of the female reproductive axis involves the rapid inhibition (< 30 min) of luteinizing hormone‐releasing hormone (LHRH) secretion from hypothalamic neurons. This fast time‐course suggests interactions with potential plasma membrane binding sites that could result in short‐term effects on LHRH neurons. Because LHRH release is calcium dependent, we have studied the acute effects of 17β‐estradiol (E2) and estradiol‐peroxidase (E‐HRP) on the elevations of intracellular calcium ([Ca2+]i) induced by acetylcholine (ACh) in LHRH‐producing GT1‐7 cells. Exposure to ACh (1–100 µm) induced transient increases of [Ca2+]i, whereas pretreatment with E2 or E‐HRP (10 nm) for 2 min reduced this response by 50–60%. The effect was specific for E2 as neither 17α‐estradiol (1 µm) nor the synthetic antiestrogens ICI182 780 (1 µm) or tamoxifen (1 µm) elicited any change on the ACh‐induced Ca2+ signal. Both the latency of the effect and the response to the membrane impermeant conjugate suggested a membrane‐mediated mechanism. Such membrane binding sites for E2 in GT1‐7 cells were demonstrated by visualizing the binding of E‐HRP and estradiol‐BSA‐fluorescein isothiocyanate (E‐BSA‐FITC) conjugates. Competition studies showed that E‐HRP binding was blocked by preincubation with E2, but not with 17α‐E2, ICI182 780, tamoxifen or progesterone, indicating that the plasma membrane binding site is highly specific for E2 and exhibits a pharmacological profile different from classical estrogen receptors. We conclude that ACh‐induced increase in [Ca2+]i in GT1‐7 cells is modulated acutely by physiological E2 concentrations in a manner which is compatible with the existence of an estrogen‐specific membrane binding site.

Voltage-dependent anion channel as a resident protein of lipid rafts: post-transductional regulation by estrogens and involvement in neuronal preservation against Alzheimer's disease.

J. Neurochem. (2011) 116, 820–827.

Modulation of Aβ-induced neurotoxicity by estrogen receptor alpha and other associated proteins in lipid rafts

Some evidences have demonstrated the participation of estrogen receptors (ERs) in rapid, non-genomic actions of estrogen to promote neuroprotection against different toxic agents. However, there is still very little information about the structural nature of these receptors and the manner these proteins may be integrated into the plasma membrane. One of the plausible possibilities is that they may be localized in lipid rafts microstructures where they would be associated with other, still unknown, molecules which may modulate their physiological activities related to cell survival. In this work, we have identified in caveolar fractions of murine septal and hippocampal neurons a membrane-related ER shown to physically interact with, both, a voltage-dependent anion channel and scaffold protein caveolin-1.

On the influence of the culture conditions in bacterial antifouling bioassays and biofilm properties: Shewanella algae, a case study

BackgroundA variety of conditions (culture media, inocula, incubation temperatures) are employed in antifouling tests with marine bacteria. Shewanella algae was selected as model organism to evaluate the effect of these parameters on: bacterial growth, biofilm formation, the activity of model antifoulants, and the development and nanomechanical properties of the biofilms.The main objectives were:1) To highlight and quantify the effect of these conditions on relevant parameters for antifouling studies: biofilm morphology, thickness, roughness, surface coverage, elasticity and adhesion forces.2) To establish and characterise in detail a biofilm model with a relevant marine strain.ResultsBoth the medium and the temperature significantly influenced the total cell densities and biofilm biomasses in 24-hour cultures. Likewise, the IC50 of three antifouling standards (TBTO, tralopyril and zinc pyrithione) was significantly affected by the medium and the initial cell density. Four media (Marine Broth, MB; 2% NaCl Mueller-Hinton Broth, MH2; Luria Marine Broth, LMB; and Supplemented Artificial Seawater, SASW) were selected to explore their effect on the morphological and nanomechanical properties of 24-h biofilms. Two biofilm growth patterns were observed: a clear trend to vertical development, with varying thickness and surface coverage in MB, LMB and SASW, and a horizontal, relatively thin film in MH2. The Atomic Force Microscopy analysis showed the lowest Young modulii for MB (0.16 ± 0.10 MPa), followed by SASW (0.19 ± 0.09 MPa), LMB (0.22 ± 0.13 MPa) and MH2 (0.34 ± 0.16 MPa). Adhesion forces followed an inverted trend, being higher in MB (1.33 ± 0.38 nN) and lower in MH2 (0.73 ± 0.29 nN).ConclusionsAll the parameters significantly affected the ability of S. algae to grow and form biofilms, as well as the activity of antifouling molecules. A detailed study has been carried out in order to establish a biofilm model for further assays. The morphology and nanomechanics of S. algae biofilms were markedly influenced by the nutritional environments in which they were developed. As strategies for biofilm formation inhibition and biofilm detachment are of particular interest in antifouling research, the present findings also highlight the need for a careful selection of the assay conditions.

An ICI 182,780-Sensitive, Membrane-Related Estrogen Receptor Contributes to Estrogenic Neuroprotective Actions against Amyloid-Beta Toxicity

Abstract: Although estrogen (E2)‐related neuroprotection has been repeatedly demonstrated in different models, the involvement of non‐classical estrogen receptors (ERs) in this activity remains unclear. Using SN56 murine cholinergic cell line from the basal forebrain, we present evidence indicating that an ER associated with the plasma membrane participates in estrogen‐dependent reduction of neuronal death induced by amyloid‐β peptide (Aβ) toxicity. Exposure to either E2 or estradiol‐horseradish peroxidase (E‐HRP) for 15 min significantly reduced Aβ‐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 neighboring the ligand‐binding domain of ERa. Using MC‐20 antibody in unpermeabilized SN56 cells, we detected a protein at the plasma membrane region. The binding of impermeant forms of E2, E‐HRP, and E‐BSA‐FITC to specific sites of SN56 plasma membrane was blocked by pre‐incubation with E2, ICI 182,780, and MC‐20 antibody in a concentration‐dependent manner. Thus, a membrane‐related ER that shares some structural homologies with ERα may participate in estrogen‐mediated neuroprotection.

Rapid modulatory effect of estradiol on acetylcholine-induced Ca2+ signal is mediated through cyclic-GMP cascade in LHRH-releasing GT1-7 cells.

Hypothalamic luteinizing hormone‐releasing hormone neurons (LHRH) form the final pathway for the central control of reproduction through the release of LHRH into the pituitary‐hypothalamic system. We previously found that LHRH‐producing GT1‐7 cells respond to acetylcholine (ACh) with an increase in intracellular calcium ([Ca2+]i) through activation of muscarinic receptors. This effect is acutely modulated by 17β‐estradiol in a manner compatible with specific membrane binding sites. Because increasing evidence suggests that second messengers are involved in the rapid action of estradiol, the aim of the present study was to identify the pathway underlying estrogen actions on ACh‐induced Ca2+ signals. 8‐Bromoguanosine 3′,5′‐cyclic monophosphate (10 µm) and C‐type natriuretic peptide (10 µm) mimicked the effect of estradiol. On the contrary, neither dibutyryl cAMP (100 µm), forskolin (100 nm or 10 µm), or sodium nitroprusside (10 µm) induced any modification of [Ca2+]i in response to ACh. The effect of estradiol on calcium transients was totally blocked by two different cGMP‐dependent protein kinase (PKG) inhibitors. In addition, phosphorylation of inositol 1,4,5‐triphosphate (IP3) receptor was rapidly induced by estradiol but totally blocked when the cells were pretreated with a PKG inhibitor. We conclude that physiological concentrations of estradiol reduce ACh‐induced Ca2+ transients via a mechanism involving a membrane‐associated guanylate cyclase, which finally induces a PKG‐dependent IP3 receptor phosphorylation that modifies calcium release from the endoplasmic reticulum.

Unique SERM-like properties of the novel fluorescent tamoxifen derivative FLTX1.

Tamoxifen is a selective estrogen receptor modulator extensively used on estrogen receptor-positive breast cancer treatment. However, clinical evidences demonstrate the increased incidence of undesirable side effects during chronic therapies, the most life threatening being uterine cancers. Some of these effects are related to tissue-dependent estrogenic actions of tamoxifen, but the exact mechanisms remain poorly understood. We have designed and synthesized a novel fluorescent tamoxifen derivative, FLTX1, and characterized its biological and pharmacological activities. Using confocal microscopy, we demonstrate that FLTX1 colocalizes with estrogen receptor α (ERα). Competition studies showed that FLTX1 binding was totally displaced by unlabeled tamoxifen and partially by estradiol, indicating the existence of non-ER-related triphenylethylene-binding sites. Ligand binding assays showed that FLTX1 exhibits similar affinity for ER than tamoxifen. FLTX1 exhibited antiestrogenic activity comparable to tamoxifen in MCF7 and T47D cells transfected with 3xERE-luciferase reporter. Interestingly, FLTX1 lacked the strong agonistic effect of tamoxifen on ERα-dependent transcriptional activity. Additionally, in vivo assays in mice revealed that unlike tamoxifen, FLTX1 was devoid of estrogenic uterotrophic effects, lacked of hyperplasic and hypertrophic effects, and failed to alter basal proliferating cell nuclear antigen immunoreactivity. In the rat uterine model of estrogenicity/antiestrogenicity, FLTX1 displayed antagonistic activity comparable to tamoxifen at lower doses, and only estrogenic uterotrophy at the highest dose. We conclude that the fluorescent derivative FLTX1 is not only a suitable probe for studies on the molecular pharmacology of tamoxifen, but also a potential therapeutic substitute to tamoxifen, endowed with potent antiestrogenic properties but devoid of uterine estrogenicity.

Expression and localization of the immunophilin FKBP51 in colorectal carcinomas and primary metastases, and alterations following oxaliplatin-based chemotherapy

The immunophilin FK506-binding protein 5 (FKBP51) is a scaffold protein that serves a pivotal role in the regulation of multiple signaling pathways, integrating external and internal stimuli into distinct signal outputs. In a previous study, we identified several genes that are significantly up- or downregulated in the peripheral white cells (PWCs) of colorectal adenocarcinoma (CRC) patients undergoing oxaliplatin-based chemotherapy. In our screening, FKBP51 gene expression was downregulated following chemotherapy. In order to determine whether this alteration in gene expression observed in PWCs may be detected at the protein level in tumors and metastases following the administration of adjuvant chemotherapy, an immunohistochemical analysis of FKBP51 in CRC and primary metastasis tissues was performed. The present study confirmed the downregulation of FKBP51 gene expression elicited by chemotherapy with folinic acid (leucovorin), fluorouracil and oxaliplatin in metastasized liver tissue that had been resected after the oxaliplatin-based chemotherapy, compared with tissue section samples of CRC from patients (prior to antineoplastic treatment). Furthermore, the results indicated that, in CRC tissue sections, the expression of FKBP51 protein is associated with an immature phenotype of stromal fibroblasts and with the epithelial-to-mesenchymal transition (EMT) phenotype, suggesting a role for this protein in the EMT process in CRC. Finally, the observation that only certain cells of the stroma express FKBP51 protein suggests a potential role for this immunophilin as a stroma cell subtype marker.

Colocalization of Estrogen Receptors with the Fluorescent Tamoxifen Derivative, FLTX1, Analyzed by Confocal Microscopy.

Tamoxifen is a selective estrogen receptor modulator that competitively binds the ligand-binding domain of estrogen receptors. Binding of tamoxifen displaces its cognate ligand, 17β-estradiol, thereby hampering the activation of estrogen receptors. Cellular labeling of ER is typically carried out using specific antibodies which require permeabilization of cells, incubation with secondary antibodies, and are expensive and time consuming. In this article, we describe the usefulness of FLTX1, a novel fluorescent tamoxifen derivative, which allows the labeling of estrogen receptors in immunocytochemistry and immunohistochemistry studies, both under permeabilized and non-permeabilized conditions. Further, besides labeling canonical estrogen receptors, this novel fluorescent probe is also suitable for the identification of unconventional targets such membrane estrogen receptors as well as other noncanonical targets, some of which are likely responsible for the number of undesired side effects reported during long-term tamoxifen treatments.