Specific inhibition of estrogen receptor alpha function by antisense oligodeoxyribonucleotides

We have tested the effect of a range of antisense oligodeoxyribonucleotides (ODN) directed against the human estrogen receptor alpha (ERalpha) on ERalpha protein expression and function. Antisense ERalpha ODN transfected into the ERalpha-positive human breast carcinoma cell line MCF7-K2 showed variable responses dependent on the oligo used. The most active antisense ODN (oligo 7) decreased the levels of ERa protein by 61% as measured by Western blot analysis. Exogenous 17beta-estradiol (17beta-E2), but not 17alpha-E2, augmented this effect, with a threshold effect at 10(-8) M 17beta-E2. The inhibitory effect of antisense ERa oligo 7 was confirmed by measurement of functional ERalpha protein. 3H-17beta-E2 binding to MCF7 cell extracts was inhibited to approximately 40% of control values in the presence of oligo 7. Antisense-transfected MCF7-K2 cell cultures produced a further 30% binding reduction in the presence of exogenous 17beta-E2. An inhibitory effect on 17beta-E2-dependent cell function was confirmed by the demonstration that ERalpha oligo 7-transfected MCF7-K2 cells failed to exhibit 17beta-E2-stimulated cell proliferation. Exogenous 17beta-E2 enhanced the inhibitory effect of the antisense ODN by increasing ODN transfection efficiency but without ERalpha catabolism via the proteosomal pathway, suggesting an effect of 17beta-E2 on the plasma membrane and the existence of different ERalpha degradation pathways in the MCF7-K2 cell subclone. As 17beta-E2 had no effect on ERalpha protein degradation, we conclude that the observed reduction of ERalpha protein levels is due solely to the presence of the antisense ERalpha ODN. Antisense ERalpha ODN molecules, therefore, may form the basis of effective therapies against ERalpha-dependent malignancies.     

Requirements for estrogen receptor alpha membrane localization and function

The estrogen receptor alpha (ERalpha) exists as a functional receptor at the plasma membrane. The structural requirements for localization and function are not well understood. Several laboratories have recently elucidated certain requirements. We recently found the translocation of ERalpha to the membrane in the absence of estrogen is dependent on caveolin-1 and serine 522 of the ERalpha protein. Mutation of serine 522 to alanine results in a 62% decrease in membrane localization and association with caveolin-1. Similarly, deletion of the caveolin-1 scaffolding domain (amino acids 60-100) largely prevents the localization of ERalpha at the plasma membrane. In the presence of estradiol (E2), ERalpha, Src-homology and collagen homology (Shc), and insulin-like growth factor receptor-1 proteins associate with and increase the localization of ERalpha at the membrane. Membrane-localized ERalpha functions as an atypical G-protein coupled receptor. There is no good evidence that ERalpha spans the membrane or contains an extracellular domain. E2/ERalpha activates different G-proteins in cell context-related fashion. These G-proteins lead to the activation of Src through PLC, PKC, IP3 and calcium influx. In breast cancer, Src activates matrix metalloproteinase-2 and -9, which cleaves heparin binding epidermal growth factor, and thus activates EGFR. This leads to downstream signaling through ERK and PI3 kinase, imparting cell growth and survival.     

Modulation of Abeta-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.     

DBC-1 mediates endocrine resistant breast cancer cell survival

No abstract available.     

Inhibition of estrogen receptor alpha expression and function in MCF-7 cells by kaempferol

Estrogens are mitogenic for estrogen receptor (ER)-positive breast cancer cells. Current treatment of ER-positive breast tumors is directed towards interruption of estrogen activity. We report that treatment of ER-positive breast cancer cells with kaempferol resulted in a time- and dose-dependent decrease in cell number. The concentration required to produce 50% growth inhibition at 48 h was approximately 35.0 and 70.0 microM for ER-positive and ER-negative breast cancer cells, respectively. For MCF-7 cells, a reduction in the ER-alpha mRNA equivalent to 50, 12, 10% of controls was observed 24 h after treatment with 17.5, 35.0, and 70.0 microM of kaempferol, respectively. Concomitantly, these treatments led to a 58, 80, and 85% decrease in ER-alpha protein. The inhibitory effect of kaempferol on ER-alpha levels was seen as early as 6 h post-treatment. Kaempferol treatment also led in a dose-dependent decrease in the expression of progesterone receptor (PgR), cyclin D1, and insulin receptor substrate 1 (IRS-1). Immunocytochemical study revealed that ER-alpha protein in kaempferol-treated MCF-7 cells formed an aggregation in the nuclei. Kaempferol also induced degradation of ER-alpha by a different pathway than that were observed for the antiestrogen ICI 182,780 and estradiol. Estradiol-induced MCF-7 cell proliferation and expression of the estrogen-responsive-element-reporter gene activity were abolished in cells co-treated with kaempferol. These findings suggest that modulation of ER-alpha expression and function by kaempferol may be, in part, responsible for its anti-proliferative effects seen in in vitro.     

Modulation of hypothalamic NMDA receptor function by cyclic AMP-dependent protein kinase and phosphatases

In the present study we investigated the modulation of hypothalamic NMDA receptor-mediated currents by cyclic AMP-dependent protein kinase (PKA) using the two-electrode voltage-clamp technique in XENOPUS: oocytes injected with rat hypothalamic mRNA. Application of forskolin, which activates PKA by means of cyclic AMP stimulation, caused a transient increase of NMDA-induced currents, whereas the inactive forskolin analogue 1,9-dideoxyforskolin had no effect. Incubation of oocytes with a membrane-permeable analogue of cyclic AMP, 8-bromoadenosine 3',5' -cyclic monophosphate, potentiated NMDA responses even more prominently than with forskolin. NMDA-induced currents recorded from XENOPUS: oocytes injected with cRNA encoding the NMDA receptor subunits NR1, NR2A, and/or NR2B, mainly found in rat hypothalamus, were not affected by PKA activation but were increased by protein kinase C (PKC) stimulation. It is interesting that inhibition of endogenous protein phosphatase 1 and/or 2A by calyculin A resulted in a similar enhancement of hypothalamic NMDA-induced currents. Preinjection of oocytes with calyculin A impeded the PKA- but not the PKC-mediated potentiation of hypothalamic NMDA-induced currents. We propose the involvement of an additional third messenger in the PKA effect, which acts most likely via the inhibition of tonically active protein phosphatase 1 and/or 2A.     

Estrogen regulation of trefoil factor 1 expression by estrogen receptor alpha and Sp proteins

Estrogen-responsive genes in human breast cancer cells often have an estrogen response element (ERE) positioned next to an Sp1 binding site. In chromatin immunoprecipitation (ChIP) assays, we investigated the binding of estrogen receptor alpha (ER), Sp1, and Sp3 to the episomal and native estrogen-responsive trefoil factor 1 (TFF1; formerly pS2) promoter in MCF-7 breast cancer cells. Mutation of the Sp site upstream of the ERE reduced estrogen responsiveness and prevented binding of Sp1 and Sp3, but not ER to the episomal promoter. In the absence of estradiol (E2), Sp1, Sp3, histone deacetylase 1 (HDAC), and HDAC2, and low levels of acetylated H3 and H4 are associated with the native promoter, with the histones being engaged in dynamic reversible acetylation. Following E2 addition, levels of ER and acetylated H3 and H4 bound to the native promoter increases. There is clearance of Sp1, but not of Sp3, from the promoter while HDAC1 and HDAC2 remain bound. These data are consistent with a model in which Sp1 or Sp3 aid in recruitment of HDACs and histone acetyltransferases (HATs) to mediate dynamic acetylation of histones associated with the TFF1 promoter, which is in a state of readiness to respond to events occurring following the addition of estrogen.