Regulation of leptin expression by 17beta-estradiol in human placental cells involves membrane associated estrogen receptor alpha

The placenta produces a wide number of molecules that play essential roles in the establishment and maintenance of pregnancy. In this context, leptin has emerged as an important player in reproduction. The synthesis of leptin in normal trophoblastic cells is regulated by different endogenous biochemical agents, but the regulation of placental leptin expression is still poorly understood. We have previously reported that 17β-estradiol (E(2)) up-regulates placental leptin expression. To improve the understanding of estrogen receptor mechanisms in regulating leptin gene expression, in the current study we examined the effect of membrane-constrained E(2) conjugate, E-BSA, on leptin expression in human placental cells. We have found that leptin expression was induced by E-BSA both in BeWo cells and human placental explants, suggesting that E(2) also exerts its effects through membrane receptors. Moreover E-BSA rapidly activated different MAPKs and AKT pathways, and these pathways were involved in E(2) induced placental leptin expression. On the other hand we demonstrated the presence of ERα associated to the plasma membrane of BeWo cells. We showed that E(2) genomic and nongenomic actions could be mediated by ERα. Supporting this idea, the downregulation of ERα level through a specific siRNA, decreased E-BSA effects on leptin expression. Taken together, these results provide new evidence of the mechanisms whereby E(2) regulates leptin expression in placenta and support the importance of leptin in placental physiology.     

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.     

Pancreatic insulin content regulation by the estrogen receptor ER alpha

The function of pancreatic beta-cells is the synthesis and release of insulin, the main hormone involved in blood glucose homeostasis. Estrogen receptors, ER alpha and ER beta, are important molecules involved in glucose metabolism, yet their role in pancreatic beta-cell physiology is still greatly unknown. In this report we show that both ER alpha and ER beta are present in pancreatic beta-cells. Long term exposure to physiological concentrations of 17beta-estradiol (E2) increased beta-cell insulin content, insulin gene expression and insulin release, yet pancreatic beta-cell mass was unaltered. The up-regulation of pancreatic beta-cell insulin content was imitated by environmentally relevant doses of the widespread endocrine disruptor Bisphenol-A (BPA). The use of ER alpha and ER beta agonists as well as ER alphaKO and ER betaKO mice suggests that the estrogen receptor involved is ER alpha. The up-regulation of pancreatic insulin content by ER alpha activation involves ERK1/2. These data may be important to explain the actions of E2 and environmental estrogens in endocrine pancreatic function and blood glucose homeostasis.     

Aggression and arginine vasopressin immunoreactivity regulation by androgen receptor and estrogen receptor alpha

In the following study, we asked which steroid receptors regulate aggression and arginine vasopressin (AVP) immunoreactivity (– ir) in several limbic regions. Using spontaneous mutant and knockout mice, we generated a novel cross of mice whose offspring lacked estrogen receptor α (ERα), androgen receptor (AR) or both ERα and AR. The wild-type (WT) males and females were compared with ERα knockout (ERαKO) male, mutated AR (Tfm) male and ERαKO/Tfm (double knockout; DKO) male littermates. Animals were gonadectomized and treated with 17β-estradiol (E2) prior to resident-intruder aggression tests. WT and Tfm males showed aggression whereas WT females, ERαKO and DKO males did not. In the lateral septum, WT and Tfm male brains had significantly denser AVP-ir as compared with WT females and DKO males. ERαKO male brains were intermediate in the amount of AVP-ir present. In the medial amygdala, brains from all genotypes had equivalent AVP-ir, except DKO males, which had significantly less AVP-ir. Overall, the expression of aggressive behavior coincided with AVP-ir in WT, Tfm and DKO males. However, in ERαKO males and WT females, the amount of AVP-ir was not associated with resident-intruder aggression. In sum we have shown that E2 acts via ERα to regulate aggression in male mice. In contrast both ERα and AR contribute to AVP-ir in limbic brain regions.     

Ligands specify estrogen receptor alpha nuclear localization and degradation

Background  

The estrogen receptor alpha (ERα) is found predominately in the nucleus, both in hormone stimulated and untreated cells. Intracellular distribution of the ERα changes in the presence of agonists but the impact of different antiestrogens on the fate of ERα is a matter of debate.     

Stimulation of catecholamine synthesis through unique estrogen receptors in the bovine adrenomedullary plasma membrane by 17beta-estradiol

Incubation of cultured bovine adrenal medullary cells with 17beta-estradiol (E(2)) (0.3-100nM) or membrane-impermeable E(2)-bovine serum albumin (100nM) acutely increased (14)C-catecholamine synthesis from [(14)C]tyrosine. The stimulatory effect of E(2) was not inhibited by ICI182,780, a nuclear estrogen receptor inhibitor. E(2) also increased tyrosine hydroxylase activity and p44/42MAPK phosphorylation, the former of which was attenuated by U0126, an inhibitor of p44/42MAPK kinase. The plasma membrane isolated from the gland showed two classes of specific binding sites of [(3)H]E(2) with apparent K(d)s of 3.2 and 106nM, and B(max)s of 0.44 and 8.5pmol/mg protein, respectively. The high-affinity binding of [(3)H]E(2) was most strongly inhibited by E(2) and phytoestrogens, and to lesser extents by other steroid hormones, while it was enhanced by ICI182,780 and environmental estrogenic pollutants. These findings suggest that E(2) acutely stimulates catecholamine synthesis via activation of p44/42MAPK through unique estrogen receptors in the plasma membrane of bovine adrenal medulla.     

Vascular endothelial growth factor receptor-2 expression is down-regulated by 17beta-estradiol in MCF-7 breast cancer cells by estrogen receptor alpha/Sp proteins

17beta-Estradiol (E2) induces and represses gene expression in breast cancer cells; however, the mechanisms of gene repression are not well understood. In this study, we show that E2 decreases vascular endothelial growth factor receptor 2 (VEGFR2) mRNA levels in MCF-7 cells, and this gene was used as a model for investigating pathways associated with E2-dependent gene repression. Deletion analysis of the VEGFR2 promoter indicates that the proximal GC-rich motifs at -58 and -44 are critical for the E2-dependent decreased response in MCF-7 cells. Mutation or deletion of these GC-rich elements results in loss of hormone responsiveness and shows that the -60 to -37 region of the VEGFR2 promoter is critical for both basal and hormone-dependent decreased VEGFR2 expression in MCF-7 cells. Western blot, immunofluorescent staining, RNA interference, and EMSAs support a role for Sp proteins in hormone-dependent down-regulation of VEGFR2 in MCF-7 cells, primarily through estrogen receptor (ER)alpha/Sp1 and ERalpha/Sp3 interactions with the VEGFR2 promoter. Using chromatin immuno-precipitation and transient transfection/RNA interference assays we show that the ERalpha/Sp protein-promoter interactions are accompanied by recruitment of the co-repressors SMRT (silencing mediator of retinoid and thyroid hormone receptor) and NCoR (nuclear receptor corepressor) to the promoter and that SMRT and NCoR knockdown reverse E2-mediated down-regulation of VEGFR2 expression in MCF-7 cells. This study illustrates that both SMRT and NCoR are involved in E2-dependent repression of VEGFR2 in MCF-7 cells.     

Phenylmethylsulfonyl fluoride (PMSF) inhibits 17 beta-estradiol binding to estrogen receptor from human prostate

Estrogen receptor binding studies were performed on cytosol obtained from human benign prostatic hyperplasia (BPH) tissue. Binding assays were done in the absence or presence of various concentrations of phenylmethylsulfonyl fluoride (PMSF). Saturation analysis and Scatchard plots showed that the binding of 17 beta -estradiol to the estrogen receptor (ER) was inhibited by PMSF. The nature of the inhibition appears to be uncompetitive, as determined from double-reciprocal plots. Glycerol density gradient centrifugation analysis also confirmed the results obtained with Scatchard plots. The inhibition observed in the presence of dithiothreitol (DTT) was greater than the inhibition observed in the absence of DTT. The maximum number of binding sites (Bmax) observed in our present study was 59.1 +/- 34.1 fmol/mg protein with an equilibrium dissociation constant (KD) of 2.2 +/- 2.2 nM. Our study indicates that PMSF significantly affects 17 beta -estradiol binding to ER and consequently alters the estimation of ER in Human BPH.     

Time course of anterior pituitary estrogen receptor after low 17 beta-estradiol doses in ovariectomized rats

The present paper describes the effect of three 17-beta-estradiol (E2) doses (1, 10 and 500 ng E2/kg) on the cytosolic and nuclear estrogen receptor content of anterior pituitary (Ap) of ovariectomized rats. The estrogen receptors were measured by [3H]E2 exchange in cytosol and crude nuclear fractions. Two hours after the administration of 10 or 500 ng E2/kg the Rc showed a depletion to 20-30% of preinjection level. The 1 ng E2/kg dose did not provoke any Rc depletion. The Rc replenishment was completed 5 h after injection of 10 ng E2/kg, but it was delayed to 10 h after injection of 500 ng E2/kg. An increased amount of Rc over the control levels was produced by 1 and 10 ng E2/kg doses, but not by the 500 ng E2/kg. The Rn level in Ap increased significantly after all E2 doses, and their highest levels were similar for 1, 10 and 500 ng E2/kg. These results suggest that some estrogenic responses like synthesis of the estrogen receptor proteins, can be elicited without previous significant Rc depletion. The relationships between Rc and Rn in Ap suggest an autoregulatory mechanism for the control of the cellular level of unbound estrogen receptors, that can be altered by the exogenous E2.     

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.     

Analysis of estrogen receptor alpha signaling complex at the plasma membrane

There is accumulating evidence that the estrogen receptor (ER) can transduce specific signals at the plasma membrane. We tried to clarify the biological function of ER as a signaling molecule by identifying proteins that interact with the membrane-localized ER. The activation function 1 and 2 (AF-1 and AF-2) domains of ERalpha with or without the membrane-targeting sequence were stably expressed in the breast cancer cell line, MCF-7. The level of tyrosine phosphorylation of AF-2 was significantly elevated by the membrane localization. By mass-spectrometry analysis, alpha- and beta-tubulins and heat shock protein 70 were identified as the AF-1-associated proteins. Of these, tubulins are associated only with membrane-targeted AF-1.