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[Display omitted] • Binding free energy studies showed that MMGBSA approach reproduced better the experimental binding free energy tendency. • Per-residue decomposition analysis allowed identification of the key residues contributing the most to the binding. • Differences in affinity between ERβ and ligands result from the number and energy of the total per-residue contribution. Estrogen receptors (ERs) are nuclear factors that exist as two subtypes: ERα and ERβ. Among the different selective ERβ agonist ligands, the widely used ERβ-selective agonist DPN (diarylpropionitrile) is highlighted. Recent experimental and thermodynamic information between R-DPN and S-DPN enantiomers with ERβ is important for evaluating further the ability of MD simulations combined with end-point methods to reproduce experimental binding affinity and generate structural insight not provided through crystallographic data. In this research, starting from crystallographic data and experimental binding affinities, we explored the structural and thermodynamic basis of the molecular recognition of ERβ with DPN and derivatives through triplicate MD simulations combined with end-point methods. Conformational analysis showed some regions with the highest mobility linked to ligand association that, at the time, impacted the total protein fluctuation. Binding free energy (ΔG) analysis revealed that the Molecular Mechanics Generalized-Born Surface Area (MMGBSA) approach was able to reproduce the experimental tendency with a strong correlation (R = 0.778), whereas per-residue decomposition analysis revealed that all the systems interacted strongly with eight residues (L298, E305, L339, M340, L343, F356, H475, and L476). The comparison between theoretical studies using the MMGBSA approach with experimental results provides new insights for drug designing of new DPN derivatives. [ABSTRACT FROM AUTHOR]

Highlights • Acute estradiol administration decrease the number of AVP immunoreactive neurons in SON and PVN. • ERβ and GPER mediate the acute effects of estradiol decreasing the AVP-ir in the PVN. • GPER mediates the acute effects of estradiol decreasing the AVP-ir in the SON. Abstract The ovarian hormone 17β-estradiol is known to regulate the release, expression and immunoreactivity of arginine-vasopressin (AVP) in the supraoptic and paraventricular hypothalamic nuclei of rodents. Previous studies have shown that estrogen receptor α is involved in the effects of chronic estradiol administration on arginine-vasopressin immunoreactivity in the female rat hypothalamus. In this study we have examined the effect of an acute administration of estradiol or specific agonists for estrogen receptors α, β and G protein-coupled estrogen receptor 1 on the immunoreactivity of arginine-vasopressin in the hypothalamus of adult ovariectomized female rats. Acute estradiol administration resulted in a significant decrease in the number of arginine-vasopressin immunoreactive neurons in the supraoptic and paraventricular nuclei after 24 h. The effects of the specific estrogen receptors agonists suggest that the action of estradiol on arginine-vasopressin immunoreactivity is mediated in the supraoptic nucleus by G protein-coupled estrogen receptor 1 and in the paraventricular nucleus by both estrogen receptor β and G protein-coupled estrogen receptor 1. Thus, in contrast to previous studies on the effect of chronic estrogenic treatments, the present findings suggest that estrogen receptor β and G protein-coupled estrogen receptor 1 mediate the acute effects of estradiol on arginine-vasopressin immunoreactivity in the hypothalamus of ovariectomized rats. [ABSTRACT FROM AUTHOR]

Aims Estrogens act as key factors in prostate biology, cellular proliferation and differentiation as well as cancer development and progression. The expression of estrogen receptor (ER)-β appears to be lost during prostate cancer progression through hypermethylation mechanism. Epigenetic drugs such as 5-aza-2′-deoxycytidine (5-AZAC) and Trichostatin A (TSA) showed efficacy in restoring ERβ expression in prostate cancer cells. This study was designed to explore the potential anti-carcinogenic effects resulting from re-expressing ERβ1 using 5-AZAC and/or TSA, followed by its stimulation with Diarylpropionitrile (DPN), a selective ERβ1 agonist, in prostate cancer cell line PC-3. Main methods Cells were treated with 5-AZAC, TSA, DPN and their combination. Subsequently, they were subjected to proliferation assays, determinations of ERβ1 expression, protein levels of active caspase-3, cyclin D1, β-catenin and VEGF. Key findings Treatment with these drugs exhibited an increase in ERβ1 expression to different extents as well as active caspase-3 levels. Meanwhile, a significant reduction in cyclin D1, VEGF and β-catenin levels was achieved as compared to the vehicle control group (p < 0.05). Interestingly, the triple combination regimen led to the most prominent anti-tumor responses in terms of increased apoptosis, reduced proliferation as well as angiogenesis. Significance The results support the notion that ERβ1 acts as a tumor suppressor protein and suggest that sequential ERβ1 expression and activation can offer significant anti-tumor responses. The study highlights that the strategy of merging epigenetic and hormonal therapies may be beneficial in treating advanced prostate cancer. [ABSTRACT FROM AUTHOR]

Estrogens have been shown to rapidly (within 1 h) affect learning and memory processes, including social recognition. Both systemic and hippocampal administration of 17β-estradiol facilitate social recognition in female mice within 40 min of administration. These effects were likely mediated by estrogen receptor (ER) α and the G-protein coupled estrogen receptor (GPER), as administration of the respective receptor agonists (PPT and G-1) also facilitated social recognition on a rapid time scale. The medial amygdala has been shown to be necessary for social recognition and long-term manipulations in rats have implicated medial amygdalar ERα. As such, our objective was to investigate whether estrogens and different ERs within the medial amygdala play a role in the rapid facilitation of social recognition in female mice. 17β-estradiol, G-1, PPT, or ERβ agonist DPN was infused directly into the medial amygdala of ovariectomized female mice. Mice were then tested in a social recognition paradigm, which was completed within 40 min, thus allowing the assessment of rapid effects of treatments. 17β-estradiol (10, 25, 50, 100 nM), PPT (300 nM), DPN (150 nM), and G-1 (50 nM) each rapidly facilitated social recognition. Therefore, estrogens in the medial amygdala rapidly facilitate social recognition in female mice, and the three main estrogen receptors: ERα, ERβ, and the GPER all are involved in these effects. This research adds to a network of brain regions, including the medial amygdala and the dorsal hippocampus, that are involved in mediating the rapid estrogenic facilitation of social recognition in female mice. [ABSTRACT FROM AUTHOR]

Background: Purpose of the present study was to investigate the effects of sexual hormones on acid and pepsin output following traumatic brain injury (TBI) induction in rats. Methods: diffuse TBI was induced by Marmarou method in female rats. Rats randomly were assigned to 9 groups: intact, TBI, TBI+OVX, vehicle, estradiol (E2), progesterone (P), E2+P combination, PPT and DPN (estrogen receptor alpha and beta agonists, respectively). The acid content and pepsin levels of each gastric washout sample were measured 5 days after the TBI induction. Results: Acid output was increased in TBI group vs intact group (P<0.01). All treated groups showed a significant increase in gastric acid secretion (P<0.001) compared to OVX+TBI group. Also, the increase in gastric acid secretion in E2+ P treated rats were significantly more than E2 (P<0.05). Gastric acid secretion in OVX+TBI animals (P<0.001) treated with PPT (P<0.01) and DPN (P<0.05) significantly increased compared to OVX+TBI. The trauma induction didn't have any effect on gastric pepsin secretion levels. The OVX+TBI animals treated with P showed a significant increase in gastric pepsin secretion compared to OVX+TBI group (P<0.01). Conclusions: Ovarian hormones through direct inhibition of acid secretion (not pepsin) have protective effects against mucosal injury following TBI. Although the issue of which receptor has mediating role in inhibitory action of estrogen on acid secretion is not clear, both alpha and beta receptors are involved in the development of estrogen. [ABSTRACT FROM AUTHOR]

Sex- and species-specific patterns of estrogen receptor (ER)-α expression are established early in development, which may contribute to sexual differentiation of behavior and determine male social organization. The current study investigated the effects of ERα and ERβ activation during the second postnatal week on subsequent alloparental behavior and ERα expression in juvenile prairie voles. Male and female pups were treated daily with 17β-estradiol (E2, ERα/ERβ agonist), PPT (selective ERα agonist), DPN (selective ERβ agonist), or the oil vehicle on postnatal days (PD) 8–14. Alloparental behavior and ERα expression were examined at PD21. PPT treatment inhibited prosocial motivation in males and increased pup-directed aggression in both sexes. E2 and DPN had no apparent effect on behavior in either sex. PPT-treated males had increased ERα expression in the medial preoptic area (MPN), medial amygdala (MEApd) and bed nucleus of the stria terminalis (BSTpr). DPN treatment also increased ERα expression in males, but only in the BSTpr. Female ERα expression was unaffected by treatment. These results support the hypothesis that ERα activation in early life is associated with less prosocial patterns of central ERα expression and alloparental behavior in males. The lack of an effect of E2 on behavior suggests that ERβ may antagonize the effects of ERα on alloparental behavior. The results in DPN-treated males suggest that ERα in the MEApd, and not the BSTpr, may be a primary determinant of alloparental behavior in males. [ABSTRACT FROM AUTHOR]

Objective: To investigate the effect of the estrogen receptor-0 (ER0) agonist diarylpropionitrile (DPN) on lipopolysaccharide (LPS)-in-duced regulated on activation normal T cell expressed and secreted (RANTES) production in macrophages and the possible mechanisms. Design: Cellular and molecular biology experimental study. Setting: University-based research laboratory. Patient(s): None. Intervention(s): ER/J mRNA and protein expression determined in murine macrophage cell line RAW264.7 cells using reverse-transcription polymerase chain reaction and Western blot analysis; RANTES production detected by ELISA in LPS-stimulated RAW264.7 cells and ER/3 knockdown RAW264.7 cells after the addition of DPN, phosphorylation of p65 and kB degradation detected by Western blot analysis; and nuclear accumulation of p65 visualized using immunofluorescence. Main Outcome Measure(s): LPS-induced RANTES production and phosphorylation of p65 and kB. Result(s): ER/3 was expressed in RAW264.7 cells, and DPN statistically significantiy decreased LPS-induced RANTES production in RAW264.7 cells. Small interfering RNA targeting the ERβ gene inhibited the effect of DPN on RANTES production. In addition, DPN inhibited nuclear translocation and phosphorylation of p65 by inhibiting kB degradation and thus prohibited the activation of nuclear factor KB (NF-KB). Conclusion(s): Diarylpropionitrile down-regulates LPS-induced RANTES production via ERβ This effect of DPN is likely due to repression of nuclear factor kB activation. [ABSTRACT FROM AUTHOR]

Objectives: The purpose of this study was to evaluate the role of female sexual hormones on the intestinal NF-κB levels following traumatic brain injury (TBI) induction. Materials and methods: Diffuse TBI was induced by Marmarou method in ovariectomized (OVX) female rats. Rats randomly assigned into 10 groups: intact, sham+OVX, TBI, TBI+OVX, vehicle, estradiol (E2), progesterone (P), E2+P, PPT and DPN (estrogen receptor alpha and beta agonists, respectively). The intestinal NF-κB p50 and p65 levels were measured by ELISA method 5 days after TBI. Results: The intestinal levels of NF-κB p65 were decreased in TBI group vs intact group (P<0.001). All treatment groups had caused a significant reduction in intestinal levels of NF-κB p65 (P<0.001) vs OVX + TBI. The trauma induction doesn't have any effect on the intestinal NF-κB p50 levels. The OVX+TBI animals treated with estrogen, progesterone and estrogen+ progesterone showed a significant reduction (P<0.05) vs OVX + TBI (P<0.001). PPT had caused a significant reduction in intestinal levels of NF-κB p50 (P <0.05) compared to OVX + TBI. PPT and DPN significantly increased the intestinal levels of NF-κB p50 vs vehicle group (P <0.05). Conclusion: The binding activity of different subunits of NF-κB (p50 and p65) varies following traumatic brain injury. Also, because the NF-κB binding activity reduced in intestinal tissue after treatment with steroids, it is possible that steroids do their performance by reducing the activity of NF-κB and thereby reduce the production of intestinal inflammatory cytokines. [ABSTRACT FROM AUTHOR]

Abstract: Gender-related differences in medulloblastoma (MB) development have been reported with a higher incidence in males (slightly above 60%) than in females, female gender being also a significantly favorable prognostic factor in MB. The present study focused on the evaluation of the mechanisms by which estrogens protect against MB formation. To this end, we used a well characterized mouse model of MB – the Patched1 heterozygous mice. Ovariectomized mice were treated with 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN), a highly potent ERβ agonist, or 4,4′,4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol (PPT), a highly potent ERα agonist. Our results show that the ERβ selective agonist DPN significantly inhibits development of MB preneoplastic lesions when compared with untreated ovariectomized mice, restoring the final incidence to that observed in the intact controls, and that these effects were achieved via activation of anti-proliferative and pro-apototic pathways. On the other hand, the ERα selective agonist PPT did not influence MB tumorigenesis relative to untreated ovariectomized mice. [Copyright &y& Elsevier]

Abstract: We have previously reported that human cultured bone cells (hObs) respond to estradiol-17β (E2) by stimulating DNA synthesis, creatine kinase BB specific activity (CK) and other parameters sex-specifically. We now investigate the sex specificity of the response of these hObs to estrogen receptor (ER) α and ERβ specific agonists. Real time PCR revealed that all cells express mRNA for both ERs. ERα mRNA but not ERβ mRNA was stimulated by all estrogenic compounds in both pre- and post-menopausal hObs with no effect in male hObs. Cells treated with E2, 2,3-bis (4-hydroxyphenyl)-propionitrile (DPN; ERβ specific agonist) and 4,4′,4″-[4-propyl-(1H)-pyrazol-1,3,5-triyl] tris–phenol (PPT; ERα specific agonist) showed increased DNA synthesis and CK in all female but not male hObs. Raloxifene (Ral), a specific ERα antagonist, inhibited the stimulation of DNA synthesis and CK by E2 or PPT, but not by DPN. DPN and PPT like E2 modulated the expression of both 12 and 15 lipooxygenase (LO) mRNA in both female but not male hObs. 12 and 15 HETE production was modulated only by DPN and PPT in these cells. The LO inhibitor baicaleine inhibited only E2 and PPT but not DPN effects in both female hObs. In conclusion, we provide herein evidence for the separation of age- and sex-dependent mediation via both ERα and ERβ pathways in the effects of estrogens on hObs, with a yet unknown mechanism. [Copyright &y& Elsevier]

Abstract: The organization of the developing male rodent brain is profoundly influenced by endogenous steroids, most notably estrogen. This process may be disrupted by estrogenic endocrine disrupting compounds (EDCs) resulting in altered sex behavior and the capacity to attract a mate in adulthood. To better understand the relative role each estrogen receptor (ER) subtype (ERα and ERβ) plays in mediating these effects, we exposed male Long Evans rats to estradiol benzoate (EB, 10μg), vehicle, or agonists specific for ERβ (DPN, 1mg/kg) or ERα (PPT, 1mg/kg) daily for the first four days of life, and then assessed adult male reproductive behavior and attractiveness via a partner preference paradigm. DPN had a greater adverse impact than PPT on reproductive behavior, suggesting a functional role for ERβ in the organization of these male-specific behaviors. Therefore the impact of neonatal ERβ agonism was further investigated by repeating the experiment using vehicle, EB and additional DPN doses (0.5mg/kg, 1mg/kg, and 2mg/kg bw). Exposure to DPN suppressed male reproductive behavior and attractiveness in a dose dependent manner. Finally, males were exposed to EB or an environmentally relevant dose of genistein (GEN, 10mg/kg), a naturally occurring xenoestrogen, which has a higher relative binding affinity for ERβ than ERα. Sexual performance was impaired by GEN but not attractiveness. In addition to suppressing reproductive behavior and attractiveness, EB exposure significantly lowered the testis to body weight ratio, and circulating testosterone levels. DPN and GEN exposure only impaired behavior, suggesting that disrupted androgen secretion does not underlie the impairment. [Copyright &y& Elsevier]

Abstract: Like other fish species, Mozambique tilapia has three forms of estrogen receptor, ERα, ERβ1, and ERβ2. A primary function of 17β-estradiol (E2) in oviparous species is the hepatic induction of the yolk precursor protein, vitellogenin (Vg). To characterize the roles of ERs in Vg production, transactivation assays and an in vivo study were carried out utilizing agonists for mammalian ERα and ERβ, and an antagonist for mammalian ERα, propyl-pyrazole-triol (PPT), diarylpropionitrile (DPN), and methyl-piperidino-pyrazole (MPP), respectively. ERα was more sensitive and responsive to PPT than ERβ1 or ERβ2 in transactivation assays. All ER isoforms indicated equivalent responsiveness to DPN compared with E2, although sensitivity to DPN was lower. MPP exhibited antagonistic action on transactivation of all ER isoforms and reduced the E2 effect on Vg and ERα 48h post-injection. DPN increased ERα and Vg expression and plasma Vg post-injection, whereas PPT was without effect; DPN seems to stimulate Vg production through activation of ERα. The ligand binding domain of all tilapia ER forms shares only 60–65% amino acid identity with human ERα and ERβ. This, together with our results, clearly indicates that agonistic or antagonistic characteristics of PPT, DPN and MPP cannot be extrapolated from mammalian to piscine ERs. [ABSTRACT FROM AUTHOR]

Abstract: The ovarian hormone estradiol regulates the expression of arginine vasopressin gene and the release of arginine vasopressin by magnocellular hypothalamic neurons. Magnocellular neurons express estrogen receptor β and are contacted by afferent neurons that express estrogen receptor α. In this study we have assessed the effect of selective ligands for estrogen receptors to determine the subtype of estrogen receptor involved in the regulation of arginine vasopressin immunoreactivity in the supraoptic and paraventricular nuclei of ovariectomized rats. The volume fraction occupied by arginine vasopressin immunoreactive material was significantly increased in both nuclei in the animals treated with estradiol compared to the animals injected with vehicle. A similar result was obtained with an estrogen receptor α selective agonist. In contrast, the administration of an estrogen receptor β selective agonist did not significantly affect arginine vasopressin immunoreactivity. This finding suggests that estradiol may regulate arginine vasopressin levels on the supraoptic and paraventricular nuclei by acting on afferent neurons expressing estrogen receptor α. [Copyright &y& Elsevier]

Abstract: Previous work has shown that continuous estradiol replacement in young ovariectomized rats enhances acquisition of a delayed matching-to-position (DMP) T-maze task over that of ovariectomized controls. The mechanism by which estradiol confers this benefit has not been fully elucidated. This study examined the role of selective estrogen receptor agonists of ERα, ERβ, and GPR30 in the enhancement of spatial learning on a DMP task by comparing continuous estradiol replacement with continuous administration of PPT (an agonist of ERα), DPN (an agonist of ERβ), or G-1 (an agonist of GPR30) relative to gonadally intact and ovariectomized vehicle-treated controls. It was found that ovariectomy impaired acquisition on this task, whereas all ER selective agonists restored the rate of acquisition to that of gonadally intact controls. These data suggest that estradiol can work through any of several estrogen receptors to enhance the rate of acquisition on this task. [Copyright &y& Elsevier]

Previous studies have shown the oestradiol modulation of brain N-methyl-d-aspartate (NMDA) receptors composed of the NR1/2B subunits. The contribution of oestrogen receptor subtypes in this oestradiol modulation of NMDA receptors and its subunits is not known. The following experiments investigated whether an oestrogenic receptor subtype is involved in the oestradiol effect on NMDA receptor specific binding and subunit mRNA levels. Ovariectomised Sprague-Dawley rats were treated 2 days after ovariectomy for 2 weeks with 17β-oestradiol, an agonist for oestrogen receptor (ER)α 4,4′,4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT) or an agonist for ERβ 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN) and compared with control vehicle-treated ovariectomised and intact rats. Uterus weights, used as a peripheral measure of oestrogenic activity, decreased after ovariectomy and increased by oestradiol and PPT but not DPN treatment. In the hippocampal CA1 oriens and CA1 radiatum, [3H]Ro 25-6981 specific binding, a NMDA/NR2B ligand, was decreased in ovariectomised compared to intact rats and this was prevented by 17β-oestradiol or PPT but not DPN treatments; a similar pattern was observed in the CA2/3 and dentate gyrus but did not reach statistical significance. In situ hybridisation of the mRNA of the NMDA/2B subunit in the hippocampus CA1, CA2/3 and dentate gyrus showed a decrease in ovariectomised rats compared to controls and this was also prevented by 17β-oestradiol and PPT but not DPN treatments. In cingulate and prefrontal cortices, ovariectomy increased [3H]Ro 25-6981 specific binding compared to intact controls, which was corrected by 17β-oestradiol treatment but neither by PPT, nor DPN. In the cortical regions, the lack of effect of the ERα or ERβ agonist whereas 17β-oestradiol was active, suggesting that the oestradiol modulation of cortical NMDA receptors requires both ERs or that this modulation does not involve ERs. In the hippocampus, the results obtained suggest an oestrogenic genomic modulation of NMDA receptors containing the NR2B subunit, implicating an ERα. [ABSTRACT FROM AUTHOR]

Abstract: The specific roles of estrogen receptor (ER) subtypes α and β in mediating estrogen’s influences on lupus autoimmunity are unknown. Herein we found that ovariectomized NZB/NZW F1 mice treated with propyl pyrazole triol (ERα-selective agonist) had significantly shorter survival, earlier development of albuminuria, higher serum concentrations of total IgG and prolactin, increased serum levels of anti-DNA IgG3, IgG2a and IgG2b and decreased anti-DNA IgG1 level compared to vehicle controls. In contrast, diarylpropionitrile (ERβ-selective agonist) administration significantly decreased serum anti-DNA IgG2b level but did not significantly affect serum levels of other anti-DNA IgG subclasses, serum total IgG or prolactin concentration, mortality or the occurrence of albuminuria. These findings suggest that ERα activation plays the predominant and immunostimulatory role in estrogen-mediated modulation of lupus while ERβ activation appears to have a slightly immunosuppressive effect on this disease. ERα activation coincidentally increased serum prolactin concentrations and may accelerate lupus disease activity also through this mechanism. [Copyright &y& Elsevier]

Abstract: Estrogens have multiple influences on immune functions. Estrogen receptors (ERs) have two distinct subtypes – α and β. To explore the specific roles of each ER subtype in estrogen-mediated immunomodulation, we investigated the effects of ER subtype-selective agonists on immune functions in ovariectomized Balb/c mice. Treatment with ERα-selective agonist propyl pyrazole triol (PPT) caused thymic atrophy and significant changes in thymic CD4/CD8 phenotypic profile. In contrast, ERβ-selective agonist diarylpropionitrile (DPN) alone had no effect on thymic weight, cellularity or CD4/CD8 phenotype expression. When coadministered with PPT, DPN partially antagonized PPT-evoked decrease in thymic cellularity and also partially attenuated PPT-induced shifts in thymic T-cell phenotype. These results indicate that ERα plays a predominant role in estrogen-induced thymic atrophy and ERβ activation may partially down-regulate ERα-mediated effects on thymic cellularity and T-cell phenotype expression. In addition, PPT administration induced a reduction in the percentage of mature B cells in the spleen, and enhanced IFN-γ production but suppressed IL-6 production from in vitro Con A-stimulated splenocytes as estradiol (E2) did, whereas DPN treatment had no effects either alone or with PPT, suggesting ERα mediates these estrogen actions. Treatment with PPT or DPN did not augment anti-DNP antibody production after DNP-KLH immunization as E2 did, implying that not merely one ER signaling pathway is involved in mediating estrogen''s effects on specific humoral immune responses. Our study further indicates ER subtype-selective agonists provide a novel approach to explore each ER subtype-mediated immunomodulation. [Copyright &y& Elsevier]

Ca2+ uptake by mitochondria is a key element in the control of cellular Ca2+ homeostasis and Ca2+-dependent phenomena. It has been known for many years that this Ca2+ uptake is mediated by the mitochondrial Ca2+ uniporter, a specific Ca2+ channel of the inner mitochondrial membrane. We have shown previously that this channel is strongly activated by a series of natural phytoestrogenic flavonoids. We show here that several agonists and antagonists of estrogen receptors (ERs) also modulate the activity of the uniporter.The specific α-ER agonist 4,4′,4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT) was the strongest activator, increasing the rate of mitochondrial Ca2+ uptake in permeabilized HeLa cells by 10-fold at 2 μM. Consistently, PPT largely increased the histamine-induced mitochondrial [Ca2+] peak and reduced the cytosolic one.Diethylstilbestrol and 17-β-estradiol (but not 17-α-estradiol) were active at pharmacological concentrations while the β-estrogen-receptor agonist 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN) was little effective.The ER modulators tamoxifen and 4-hydroxy-tamoxifen inhibited mitochondrial Ca2+ uptake (IC50 2.5±1.5 and 2.5±1.4 μM, mean±s.d., respectively) both in the presence and in the absence of PPT, but raloxifene and the pure estrogen antagonist ICI 182,780 produced no effect.Activation by PPT was immediate and inhibition by tamoxifen or 4-hydroxy-tamoxifen required only 5 min to reach maximum.Tamoxifen did not modify mitochondrial membrane potential and PPT induced a slow mitochondrial depolarization at higher concentrations than those required to activate mitochondrial Ca2+ uptake.These results suggest that some kind of ER or related protein located in mitochondria controls the activity of the Ca2+ uniporter by a nongenomic mechanism. This novel mechanism of action of estrogen agonists and antagonists can provide a new interpretation for several previously reported effects of these compounds.British Journal of Pharmacology (2005) 145, 862–871. doi:10.1038/sj.bjp.0706265; published online 23 May 2005 [ABSTRACT FROM AUTHOR]

Estrogen receptor (ER) mediated neuroprotection has been demonstrated in both in vitro and in vivo model systems. However, the relative contribution by either ER subtype, ERα or ERβ, to estrogen-induced neuroprotection remains unresolved. To address this question, we investigated the impact of selective ER agonists for either ERα, PPT, or ERβ, DPN, to prevent neurodegeneration in cultured hippocampal neurons exposed to excitotoxic glutamate. Using three indicators of neuronal viability and survival, we demonstrated that both the ERα selective agonist PPT and the ERβ selective agonist DPN protected hippocampal neurons against glutamate-induced cell death in a dose-dependent manner, with the maximal response occurring at 100 pM. Further analyses showed that both PPT and DPN enhanced Bcl-2 expression in hippocampal neurons, with an efficacy comparable to their neuroprotective capacity. Collectively, the present data indicate that activation of either ERα or ERβ can promote neuroprotection in hippocampal neurons, suggesting that both receptor subtypes could be involved in estrogen neuroprotection. As ERβ is highly expressed in the brain and has little or no expression in the breast or uterus, discovery and design of ERβ selective molecules could provide a strategy for activating the beneficial effects of estrogen in the brain without activating untoward effects of estrogen in reproductive organs. [Copyright &y& Elsevier]

Most cultured cell lines require addition of serum to the medium to maintain their proliferative capacity. For studies examining the cellular effects of estrogens serum is charcoal-stripped to remove steroids. Nonetheless, addition of the selective estrogen receptor modulator (SERM) 4-hydroxytamoxifen (4-OHT) inhibits the basal transcriptional activity of estrogen receptors alpha or beta (ERα or ERβ) in transfected cells. We tested the hypothesis that elimination of serum from the culture medium will block 4-OHT’s repression of basal activity. Chinese hamster ovary (CHO-K1) cells adapted to serum-free medium exhibited estrogen responsiveness that was identical with that of the cells grown in serum-containing media. 4-OHT-suppressed basal transcription of an estrogen response element (ERE)-reporter in ERα-transfected cells even in the absence of serum, indicating that the 4-OHT suppressive activity is not mediated by blocking ER interaction with serum estrogens. We speculate that 4-OHT-ER recruits co-repressors to suppress basal transcription. We discovered that CHO-K1 cells express ERα and ERβ mRNA. However only ERβ protein was expressed and use of ERβ-selective 2,3-bis(4-hydroxy-phenyl)propionitrile (DPN) and ERα-selective 4-propyl-1,3,5-tris(4-hydroxy-phenyl)pyrazole) (PPT) revealed that only ERβ was transcriptionally active. In conclusion, growing CHO-K1 in serum-free medium does not impact the estrogen responsiveness and this cell line expresses functional ERβ. [Copyright &y& Elsevier]

Estrogens play a key role in the sexual differentiation of the brain and behavior. While early estrogen actions exert masculinizing effects on the brain of male rodents, a diametrically opposite effect is observed in birds where estrogens demasculinize the brain of females. Yet, the two vertebrate classes express similar sex differences in the brain and behavior. Although ERα is thought to play a major role in these processes in rodents, the role of ERβ is still controversial. In birds, the identity of the estrogen receptor(s) underlying the demasculinization of the female brain remains unclear. The aim of the present study was thus to determine in Japanese quail the effects of specific agonists of ERα (propylpyrazole triol, PPT) and ERβ (diarylpropionitrile, DPN) administered at the beginning of the sensitive period (embryonic day 7, E7) on the sexual differentiation of male sexual behavior and on the density of vasotocin-immunoreactive (VT-ir) fibers, a known marker of the organizational action of estrogens on the quail brain. We demonstrate that estradiol benzoate and the ERβ agonist (DPN) demasculinize male sexual behavior and decrease the density of VT-ir fibers in the medial preoptic nucleus and the bed nucleus of the stria terminalis, while PPT has no effect on these measures. These results clearly indicate that ERβ, but not ERα, is involved in the estrogen-induced sexual differentiation of brain and sexual behavior in quail. • Quail eggs were injected at embryonic day 7 with an ERα or ERβ agonist. • The ERβ agonist DPN demasculinized copulatory behavior of adult males. • DPN reduced the density of sexually dimorphic VT-ir fibers in the preoptic area. • The ERα agonist PPT did not have any of these effects. • Quail demascuilinization is mediated by ERβ with no major contribution of ERα. [ABSTRACT FROM AUTHOR]