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.     

Increased rates of fatty acid uptake and plasmalemmal fatty acid transporters in obese Zucker rats

Giant vesicles were used to study the rates of uptake of long-chain fatty acids by heart, skeletal muscle, and adipose tissue of obese and lean Zucker rats. With obesity there was an increase in vesicular fatty acid uptake of 1.8-fold in heart, muscle and adipose tissue. In some tissues only fatty acid translocase (FAT) mRNA (heart, +37%; adipose, +80%) and fatty acid-binding protein (FABPpm) mRNA (heart, +148%; adipose, +196%) were increased. At the protein level FABPpm expression was not changed in any tissues except muscle (+14%), and FAT/CD36 protein content was altered slightly in adipose tissue (+26%). In marked contrast, the plasma membrane FAT/CD36 protein was increased in heart (+60%), muscle (+80%), and adipose tissue (+50%). The plasma membrane FABPpm was altered only in heart (+50%) and adipose tissues (+70%). Thus, in obesity, alterations in fatty acid transport in metabolically important tissues are not associated with changes in fatty acid transporter mRNAs or altered fatty acid transport protein expression but with their increased abundance at the plasma membrane. We speculate that in obesity fatty acid transporters are relocated from an intracellular pool to the plasma membrane in heart, muscle, and adipose tissues.     

Palmitoylation-dependent estrogen receptor alpha membrane localization: regulation by 17beta-estradiol

A fraction of the nuclear estrogen receptor alpha (ERalpha) is localized to the plasma membrane region of 17beta-estradiol (E2) target cells. We previously reported that ERalpha is a palmitoylated protein. To gain insight into the molecular mechanism of ERalpha residence at the plasma membrane, we tested both the role of palmitoylation and the impact of E2 stimulation on ERalpha membrane localization. The cancer cell lines expressing transfected or endogenous human ERalpha (HeLa and HepG2, respectively) or the ERalpha nonpalmitoylable Cys447Ala mutant transfected in HeLa cells were used as experimental models. We found that palmitoylation of ERalpha enacts ERalpha association with the plasma membrane, interaction with the membrane protein caveolin-1, and nongenomic activities, including activation of signaling pathways and cell proliferation (i.e., ERK and AKT activation, cyclin D1 promoter activity, DNA synthesis). Moreover, E2 reduces both ERalpha palmitoylation and its interaction with caveolin-1, in a time- and dose-dependent manner. These data point to the physiological role of ERalpha palmitoylation in the receptor localization to the cell membrane and in the regulation of the E2-induced cell proliferation.     

Membrane association of estrogen receptor alpha and beta influences 17beta-estradiol-mediated cancer cell proliferation

S-Palmitoylation is a widespread post-translational modification of integral and/or peripheral proteins occurring in all eukaryotic cells. The family of S-palmitoylated proteins is large and diverse and recently, estrogen receptor isoforms (ERalpha and ERbeta) belonging to the nuclear receptor superfamily have been added to the palmitoylproteoma. S-Palmitoylation allows ERalpha and ERbeta localization at the plasma membrane, where they associate with caveolin-1. Upon 17beta-estradiol (E2) stimulation, ERalpha dissociates from caveolin-1 allowing the activation of rapid signals relevant for cell proliferation. In contrast to ERalpha, E2 increases ERbeta association with caveolin-1 and activates p38 kinase and the downstream pro-apoptotic cascade (i.e., caspase-3 activation and PARP cleavage). These data highlight the physiological role of palmitoylation in modulating the ERalpha and ERbeta localization at the plasma membrane and the regulation of different E2-induced non-genomic functions relevant for controlling cell proliferation.     

Leptin and its receptor are controlled by 17beta-estradiol in peripheral tissues of ovariectomized rats

It has been widely shown that there is a complex interaction between sex steroids and leptin effects on body weight. In this sense, the absence of female sex steroids is linked to a significant increase in body weight, which seems to be related to an impairment of the central actions of leptin. The present study was designed to elucidate the effects of two different treatments with 17beta-estradiol on leptin receptor and serum leptin levels in ovariectomized rats, a model of postmenopausal condition. Our results have shown that plasma leptin levels in ovariectomized rats were lower than in estradiol-treated animals, thereby supporting a positive effect of this steroid. Recent information has extended leptin actions to peripheral tissues, mainly to insulin-dependent tissues, this effect being related to metabolic actions. To better understand the peripheral effects of leptin and their possible regulation by estradiol treatment, we have analyzed leptin receptor expression in the skeletal muscle and the adipose tissue. Our results showed a tissue-specific regulation of this protein: Ob-Rb expression in the adipose tissue decreased when the time of treatment or the dose of estradiol administered increased, suggesting less sensitivity to leptin in this tissue, whereas in the skeletal muscle the changes in this protein followed the same profile as the plasma leptin levels. We think that this specific regulation could ensure a different response of each tissue toward the same serum leptin level. Further studies to clarify this situation are ongoing.     

Comparisons of estradiol,LH and FSH patterns in pregnant and nonpregnant beagle bitches

To characterize plasma estradiol, LH and FSH patterns of secretion during the bitch estrous cycle, blood samples were obtained daily from 15 days before until 135 days after the LH surge in 10 pregnant and 10 nonpregnant beagle bitches. After an initial increase between days 15 and 10 and an expected proestrous peak, estradiol concentrations increased again from days 9-12 (corresponding to cytological metestrus) from basal values observed around day 9 after the LH surge, and remained significantly elevated throughout the luteal phase both in pregnant and nonpregnant animals. Concomitantly with the end of the luteal phase, plasma concentrations of estradiol returned to basal values in both groups. During the mid- to late-luteal phase, mean basal LH secretion was significantly elevated throughout in the pregnant relative to the nonpregnant animals. However, in nonpregnant animals, pulsatility was increased and peaks of higher amplitude were observed. The plasma FSH profiles, determined by a specific homologous RIA, differed significantly between pregnant and nonpregnant bitches during the last two-thirds of the luteal phase with a mean FSH level more elevated during pregnancy. The FSH level then decreased around parturition and low concentrations during lactation period were observed. The FSH concentrations remained steady in nonpregnant luteal phases from early luteal phase through mid-anestrus. The differences in pregnant and nonpregnant LH and FSH concentrations suggest pregnancy differences in regulation of the corpus luteum. Finally, the elevated estradiol concentrations observed during the luteal phase of both pregnant and nonpregnant animals suggest that an ovarian production of estrogens may be involved in overall corpus luteum regulation in dogs as in other species.     

Effect of high-fat diet on the expression of proteins in muscle, adipose tissues, and liver of C57BL/6 mice

In the present study, the effect of a high fat diet on the expression of proteins in insulin target tissues was analyzed using a proteomic approach. Gastrocnemius muscle, white and brown adipose tissue, and liver were taken from C57BL/6 mice either fed on a high-fat or a chow diet. Expression levels of approximately 10 000 polypeptides for all the four tissues were assessed by two-dimensional gel electrophoresis (2-DE). Computer-assisted image analysis allowed the detection of 50 significantly (p < 0.05) differentially expressed proteins between obese and lean mice. Interestingly, more than half of these proteins were detected in the brown adipose tissue. The differentially expressed proteins were identified by tandem mass spectrometry. Several stress and redox proteins were modulated in response to the high-fat diet. A key glycolytic enzyme was found to be downregulated in adipose tissues and muscle, suggesting that at elevated plasma fatty acid concentrations, fatty acids compete with glucose as an oxidative fuel source. Furthermore, in brown adipose tissue there were significant changes in mitochondrial enzymes involved in the Krebs tricarboxylic acid (TCA) cycle and in the respiratory chain in response to the high-fat diet. The brown adipose tissue is an energy-dissipating tissue. Our data suggest that the high-fat diet treated mice were increasing energy expenditure to defend against weight gain.     

Estrogen induces rapid decrease in dendritic thorns of CA3 pyramidal neurons in adult male rat hippocampus

Modulation of hippocampal synaptic plasticity by estrogen has been attracting much attention. Thorns of thorny excrescences of CA3 hippocampal neurons are post-synaptic regions whose presynaptic partners are mossy fiber terminals. Here we demonstrated the rapid effect of estradiol on the density of thorns of thorny excrescences, by imaging Lucifer Yellow-injected CA3 neurons in adult male rat hippocampal slices. The application of 1nM estradiol induced rapid decrease in the density of thorns on pyramidal neurons within 2h. The estradiol-mediated decrease in the density of thorns was blocked by CNQX (AMPA receptor antagonist) and PD98059 (MAP kinase inhibitor), but not by MK-801 (NMDA receptor antagonist). ERalpha agonist PPT induced the same suppressive effect as that induced by estradiol on the density of thorns, but ERbeta agonist DPN did not affect the density of thorns. Note that a 1nM estradiol treatment did not affect the density of spines in the stratum radiatum and stratum oriens. A search for synaptic ERalpha was performed using purified RC-19 antibody. The localization of ERalpha (67kDa) in the CA3 mossy fiber terminals and thorns was demonstrated using immunogold electron microscopy. These results imply that estradiol drives the signaling pathway including ERalpha and MAP kinase.     

Effect of food restriction on the insulin signalling pathway in rat skeletal muscle and adipose tissue

We tried to elucidate the effects of a brief and severe model of food restriction on insulin sensitivity in female rats, focusing on key proteins involved in the insulin signalling pathway in skeletal muscle and adipose tissue after 5, 10 and 15 days of food restriction. Using euglycemic clamp, we detected that food-restricted rats are significantly less sensitive to insulin action than control rats. However, the time of restriction promotes a progressive increase on insulin sensitivity. The analysis of the insulin signalling pathway showed a tissue-specific regulation of several proteins involved in insulin signalling. In skeletal muscle, insulin receptor substrate 1 and Glut4 are up-regulated at the end of the food restriction period, just the opposite of what we found in adipose tissue. In conclusion, a 50% reduction of food intake modulates insulin sensitivity through a tissue-specific regulation of the insulin signalling pathway in the main target tissues for this hormone.     

Regulation of cyclic adenosine 3',5'-monophosphate signaling and pulsatile neurosecretion by Gi-coupled plasma membrane estrogen receptors in immortalized gonadotropin-releasing hormone neurons

Immortalized GnRH neurons (GT1-7) express receptors for estrogen [estrogen receptor-alpha and -beta(ERalpha and ERbeta)] and progesterone (progesterone receptor A) and exhibit positive immunostaining for both intracellular and plasma membrane ERs. Exposure of GT1-7 cells to picomolar estradiol concentrations for 5-60 min caused rapid, sustained, and dose-dependent inhibition of cAMP production. In contrast, treatment with nanomolar estradiol concentrations for 60 min increased cAMP production. The inhibitory and stimulatory actions of estradiol on cAMP formation were abolished by the ER antagonist, ICI 182,780. The estradiol-induced inhibition of cAMP production was prevented by treatment with pertussis toxin, consistent with coupling of the plasma membrane ER to an inhibitory G protein. Coimmunoprecipitation studies demonstrated an estradiol-regulated stimulatory interaction between ERalpha and Galphai3 that was prevented by the ER antagonist, ICI 182,780. Exposure of perifused GT1-7 cells and hypothalamic neurons to picomolar estradiol levels increased the GnRH peak interval, shortened peak duration, and increased peak amplitude. These findings indicate that occupancy of the plasma membrane-associated ERs expressed in GT1-7 neurons by physiological estradiol levels causes activation of a Gi protein and modulates cAMP signaling and neuropeptide secretion.     

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.     

Identification and characterization of membrane estrogen receptor from MCF7 estrogen-target cells

Estrogens control the proliferation of estrogen-target cells through a receptor mediated pathway. We have recently presented evidence that estradiol cancels the proliferative inhibition exerted by albumin on estrogen-target cells (indirect-negative hypothesis). We postulate that this mechanism requires the presence of a membrane estrogen receptor (mER)-membrane albumin receptor complex. Confirmation for mERalpha in MCF7 cells is now made using both the C542 monoclonal and ER-21 polyclonal antibodies (Ab)s specific for ERalpha. Western blot analysis of purified membrane proteins with ERalpha Abs revealed multiple high M(r) mERs (92 k, 110 k, and 130 k M(r)), as well as a 67 k M(r) mER; immunoreactive proteins were competed by inclusion of 500-fold molar excess C542 peptide. Ligand blot analysis of similar extracts with estradiol-peroxidase identified several potential mERs as well; two of these proteins were also recognized by C542 and ER-21 Abs (110 and 67 k M(r)). Fluorescence, confocal and electron microscopy of MCF7 cells fixed in 2.0% paraformaldehyde/0.1% glutaraldehyde identified specific mERalpha sites by immunocytochemistry. Specific binding of 3H-17beta-estradiol was reduced by a 200-fold molar excess of unlabeled 17beta-estradiol, but not by testosterone and progesterone. These results suggest that the ER on the plasma membrane of MCF7 cells is similar, but not identical to its intracellular counterpart. We propose that the observed mER actively participates in the estrogen-mediated proliferation of MCF7 cells.     

Augmentation and suppression of action potentials by estradiol in the myometrium of pregnant rat.

The purpose of this study was to investigate the actions of estradiol on spontaneous and evoked action potentials in the isolated longitudinal smooth muscle cells of the pregnant rat. Single cells were obtained by enzymatic digestion from pregnant rat longitudinal myometrium. Action potentials and currents were recorded by whole-cell current-clamp and voltage-clamp methods, respectively. The acute effects of 17beta-estradiol on action potentials and inward and outward currents were investigated. The following results were obtained. The average resting membrane potential of single myometrial cells was -54 mV (n = 40). In many cells, an electrical stimulation evoked a membrane depolarization, and action potentials were superimposed on the depolarization. In some cells, spontaneous action potentials were observed. Estradiol (30 microM) slightly depolarized the membrane (ca. 5 mV) and attenuated the generation of action potentials by reducing the frequency and amplitude of the spikes. Afterhyperpolarization was also attenuated by estradiol (30 microM). On the other hand, in 5 of 35 cells, estradiol increased the first spike amplitude and action potential duration, while frequency of the spike generation and afterhyperpolarization were inhibited. In voltage-clamped muscle cells, estradiol inhibited both inward and outward currents. Acute inhibition or augmentation of spike generation by estradiol is due to the balance of inhibition of inward and outward currents. Inhibition of both currents also prevented afterhyperpolarization, causing potential-dependent block of Ca spikes.