ERβ Agonist Research Articles

Distinct role of estrogen receptor-alpha and beta on postmenopausal diabetes-induced vascular dysfunction

Estrogen is known to influence vascular functions and insulin sensitivity, but the relative contribution of estrogen receptor (ER) isoforms in postmenopausal diabetes-induced vascular dysfunction is unclear. The aim of the present study was to delineate the distinct role of estrogen receptor-α and beta β on the vascular function in ovariectomized diabetic rats. Age matched 60 female sprague dawley rats (200–250g) were divided in nine groups. Bilateral ovariectomy was performed and streptozotocin was used to induce experimental diabetes. Rats were administered with 10μg/kg; s.c. of a nonselective estrogen receptor agonist, 17-β estradiol (E2), selective ER-α agonist (4,4′,4″-(4-propyl-[1H] pyrazole-1,3,5-triyl) tris phenol (PPT) and selective ER-β agonist, 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN) for 4weeks after STZ injection. Treatment with selective ER-α agonist and E2 improved the impaired glycemic and lipid profile in ovariectomized diabetic rats, however selective ER-β agonist did not show any effect. Vascular endothelial dysfunction was assessed by acetylcholine and sodium nitroprusside-induced endothelium dependent and independent relaxation in isolated rat aortic ring preparation as well as by electron microscopy of thoracic aorta. Further, serum thiobarbituric acid reactive substances, tumour necrotic factor-alpha and interleukin-1 beta and C-reactive protein were estimated to assess oxidative stress and vascular inflammation. Treatment with ER-α agonist markedly and E2 partially improved vascular function and endothelial integrity along with reduction in serum TBARS and inflammatory cytokines. However, ER-β agonist did not show any improvement in vascular functions, oxidative stress or inflammation. These findings suggest that selective targeting of ER-α receptors results in vasculoprotection in the state of hypoestrogenicity and diabetes.

Oestradiol-induced synapse formation in the female hippocampus: roles of oestrogen receptor subtypes.

During the oestrus cycle, varying spine synapse density correlates positively with varying local synthesis of oestradiol in the hippocampus. In this context, the roles of the oestrogen receptor (ER) subtypes ERα and β are not fully understood. In the present study, we used neonatal hippocampal slice cultures from female rats because these cultures synthesise oestradiol and express both receptor subtypes, and inhibition of oestradiol synthesis in these cultures results in spine synapse loss. Using electron microscopy, we tested the effects on spine synapse density in response to agonists of both ERα and ERβ. Application of agonists to the cultures had no effect. After inhibition of oestradiol synthesis, however, agonists of ERα induced spine synapse formation, whereas ERβ agonists led to a reduction in spine synapse density in the CA1 region of these cultures. Consistently, up-regulation of ERβ in the hippocampus of adult female aromatase-deficient mice is paralleled by hippocampus-specific spine synapse loss in this mutant. Finally, we found an increase in spine synapses in the adult female ERβ knockout mouse, but no effect in the adult female ERα knockout mouse. Our data suggest antagonistic roles of ERβ and ERα in spine synapse formation in the female hippocampus, which may contribute to oestrus cyclicity of spine synapse density in the hippocampus.

Cell proliferation and modulation of interaction of estrogen receptors with coregulators induced by ERα and ERβ agonists

The aim of the present study was to investigate modulation of the interaction of the ERα and ERβ with coregulators in the ligand responses induced by estrogenic compounds. To this end, selective ERα and ERβ agonists were characterized for intrinsic relative potency reflected by EC50 and maximal efficacy towards ERα and ERβ mediated response in ER selective reporter gene assays, and subsequently tested for induction of cell proliferation in T47D-ERβ cells with variable ERα/ERβ ratio, and finally for ligand dependent modulation of the interaction of ERα and ERβ with coregulators using the MARCoNI assay, with 154 unique nuclear receptor coregulator peptides derived from 66 different coregulators. Results obtained reveal an important influence of the ERα/ERβ ratio and receptor selectivity of the compounds tested on induction of cell proliferation. ERα agonists activate cell proliferation whereas ERβ suppresses ERα mediated cell proliferation. The responses in the MARCoNI assay reveal that upon ERα or ERβ activation by a specific agonist, the modulation of the interaction of the ERs with coregulators is very similar indicating only a limited number of differences upon ERα or ERβ activation by a specific ligand. Differences in the modulation of the interaction of the ERs with coregulators between the different agonists were more pronounced. Based on ligand dependent differences in the modulation of the interaction of the ERs with coregulators, the MARCoNI assay was shown to be able to classify the ER agonists discriminating between different agonists for the same receptor, a characteristic not defined by the ER selective reporter gene or proliferation assays.It is concluded that the ultimate effect of the model compounds on proliferation of estrogen responsive cells depends on the intrinsic relative potency of the agonist towards ERα and ERβ and the cellular ERα/ERβ ratio whereas differences in the modulation of the interaction of the ERα and ERβ with coregulators contribute to the ligand dependent responses induced by estrogenic compounds.

Periodic Estrogen Receptor-Beta Activation: A Novel Approach to Prevent Ischemic Brain Damage.

In women, the risk for cerebral ischemia climbs rapidly after menopause. At menopause, production of ovarian hormones; i.e., progesterone and estrogen, slowly diminishes. Estrogen has been suggested to confer natural protection to premenopausal women from ischemic stroke and some of its debilitating consequences. This notion is also strongly supported by laboratory studies showing that a continuous chronic 17β-estradiol (E2; a potent estrogen) regimen protects brain from ischemic injury. However, concerns regarding the safety of the continuous intake of E2 were raised by the failed translation to the clinic. Recent studies demonstrated that repetitive periodic E2 pretreatments, in contrast to continuous E2 treatment, provided neuroprotection against cerebral ischemia in ovariectomized rats. Periodic E2 pretreatment protects hippocampal neurons through activation of estrogen receptor subtype beta (ER-β). Apart from neuroprotection, periodic activation of ER-β in ovariectomized rats significantly improves hippocampus-dependent learning and memory. Difficulties in learning and memory loss are the major consequence of ischemic brain damage. Periodic ER-β agonist pretreatment may provide pharmacological access to a protective state against ischemic stroke and its debilitating consequences. The use of ER-β-selective agonists constitutes a safer target for future research than ER-α agonist or E2, inasmuch as it lacks the ability to stimulate the proliferation of breast or endometrial tissue. In this review, we highlight ER-β signaling as a guide for future translational research to reduce cognitive decline and cerebral ischemia incidents/impact in post-menopausal women, while avoiding the side effects produced by chronic E2 treatment.

Novel ligands balance estrogen receptor β and α agonism for safe and effective suppression of the vasomotor response in the ovariectomized female rat model of menopause.

Vasomotor thermo-dysregulation (hot flashes) are an often debilitating symptom of menopause. Effective treatment is achieved primarily through activation of the estrogen receptor (ER)α with estrogens but is also associated with increased risk for breast and uterine cancer. In this study, we have tested novel compounds lacking the B ring of 17-hydroxy-β-estradiol (E2) (A-CD compounds) with differing ratios of ERα:ERβ binding affinities for the ability to reduce diurnal/nocturnal tail-skin temperatures (TSTs) in the ovariectomized female rat menopausal hot flash model. Normal mammary tissue expresses the predominantly antiproliferative ERβ. Therefore, we hypothesized that a preferential ERβ agonist with fractional ERα activity would safely reduce TSTs. The A-CD compound, L17, is a preferential ERβ agonist that has a ratio of ERβ:ERα binding affinity relative to E2 of 9.3 (where ERβ:ERα for E2, 1.0). In the ovariectomized rat, daily administration of low doses (1 mg/kg) of the A-CD compound TD81 (ERα:ERβ relative affinity, 15.2) was ineffective in temperature regulation, whereas L17 showed a trend toward TST reduction. Both E2 and the A-CD compound, TD3 (ERβ:ERα relative affinity, 5.0), also reduced TSTs but had marked proliferative effects on mammary and uterine tissues. At 2 mg/kg, L17 strongly reduced TSTs even more effectively than E2 but, importantly, had only minimal effect on uterine weight and mammary tissues. Both E2- and L17-treated rats showed similar weight reduction over the treatment period. E2 is rapidly metabolized to highly reactive quinones, and we show that L17 has 2-fold greater metabolic stability than E2. Finally, L17 and E2 similarly mediated induction of c-fos expression in neurons within the rat thermoregulatory hypothalamic median preoptic nucleus. Thus, the A-CD compound, L17, may represent a safe and effective approach to the treatment of menopausal hot flashes.

Estrogen regulates the expression of Ndrg2 in astrocytes

N-myc downstream-regulated gene 2 (Ndrg2) is a newly identified molecule that is mainly expressed in astrocytes within the central nervous system (CNS) and is involved in the proliferation and activation of astrocytes. 17β-estradiol (E2) is one of the most important circulating hormones, and in the CNS, astrocytes are a target and potential mediator of the action of E2. Our most recent study found that DPN, an estrogen receptor (ER) β-specific agonist, activated the Ndrg2 promoter and elevated endogenous NDRG2 protein expression in MCF7, HSG and T-47D cells. However, whether E2 regulates Ndrg2 expression in astrocytes remains unknown. Here, we conducted both in vivo and in vitro experiments and found that ERβ co-localized with NDRG2 in astrocytes. Furthermore, in primary cultured astrocytes, we demonstrated that E2 up-regulated Ndrg2 mRNA and protein expression in a dose- and time-dependent manner and that the ERβ agonist DPN but not the ERα agonist PPT up-regulated Ndrg2 expression. In vivo, we found that in the hippocampus of adult ovariectomized (OVX) female mice, Ndrg2 mRNA and protein expression were significantly decreased compared with those in normal adult female mice. After the OVX mice received continuous subcutaneous injections of 50μg/kg E2, 100μg/kg E2 or the ERβ agonist DPN for 10 days, the Ndrg2 expression significantly increased compared with that of the OVX mice. Our results indicate that E2 may affect astrocytes by regulating Ndrg2 expression.

Targeting estrogen receptor subtypes (ERα and ERβ) with selective ER modulators in ovarian cancer.

Ovarian cancer cells express both estrogen receptor α (ERα) and ERβ, and hormonal therapy is an attractive treatment option because of its relatively few side effects. However, estrogen was previously shown to have opposite effects in tumors expressing ERα compared with ERβ, indicating that the two receptor subtypes may have opposing effects. This may explain the modest response to nonselective estrogen inhibition in clinical practice. In this study, we aimed to investigate the effect of selectively targeting each ER subtype on ovarian cancer growth. Ovarian cancer cell lines SKOV3 and OV2008, expressing both ER subtypes, were treated with highly selective ER modulators. Sodium 3'-(1-(phenylaminocarbonyl)-3,4-tetrazolium)-bis(4-methoxy-6-nitro) benzene sulfonic acid hydrate (XTT) assay revealed that treatment with 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole dihydrochloride (MPP) (ERα antagonist) or 2,3-bis(4-hydroxy-phenyl)-propionitrile (DPN) (ERβ agonist) significantly suppressed cell growth in both cell lines. In contrast, 4,4',4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol (PPT) (ERα agonist) or 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]-pyrimidin-3-yl]phenol (PHTPP) (ERβ antagonist) significantly enhanced cell growth. These results were confirmed on a xenograft model where SKOV3 cells were injected s.c. into ovariectomized mice. We observed that the average size of xenografts in both the DPN-treated group and the MPP-treated group was significantly smaller than that for the vehicle-treated group. In addition, we found that phospho-AKT expressions in SKOV3 cells were reduced by 80% after treatment with MPP and DPN, indicating that the AKT pathway was involved. The combined treatment with MPP and DPN had a synergistic effect in suppressing ovarian cancer cell growth. Our findings indicate that targeting ER subtypes may enhance the response to hormonal treatment in women with ovarian cancer.

Multifactorial ERβ and NOTCH1 control of squamous differentiation and cancer

Downmodulation or loss-of-function mutations of the gene encoding NOTCH1 are associated with dysfunctional squamous cell differentiation and development of squamous cell carcinoma (SCC) in skin and internal organs. While NOTCH1 receptor activation has been well characterized, little is known about how NOTCH1 gene transcription is regulated. Using bioinformatics and functional screening approaches, we identified several regulators of the NOTCH1 gene in keratinocytes, with the transcription factors DLX5 and EGR3 and estrogen receptor β (ERβ) directly controlling its expression in differentiation. DLX5 and ERG3 are required for RNA polymerase II (PolII) recruitment to the NOTCH1 locus, while ERβ controls NOTCH1 transcription through RNA PolII pause release. Expression of several identified NOTCH1 regulators, including ERβ, is frequently compromised in skin, head and neck, and lung SCCs and SCC-derived cell lines. Furthermore, a keratinocyte ERβ-dependent program of gene expression is subverted in SCCs from various body sites, and there are consistent differences in mutation and gene-expression signatures of head and neck and lung SCCs in female versus male patients. Experimentally increased ERβ expression or treatment with ERβ agonists inhibited proliferation of SCC cells and promoted NOTCH1 expression and squamous differentiation both in vitro and in mouse xenotransplants. Our data identify a link between transcriptional control of NOTCH1 expression and the estrogen response in keratinocytes, with implications for differentiation therapy of squamous cancer.

Estradiol enhances object recognition memory in Swiss female mice by activating hippocampal estrogen receptor α

In rodents, 17β-estradiol (E2) enhances hippocampal function and improves performance in several memory tasks. Regarding the object recognition paradigm, E2 commonly act as a cognitive enhancer. However, the types of estrogen receptor (ER) involved, as well as the underlying molecular mechanisms are still under investigation. In the present study, we asked whether E2 enhances object recognition memory by activating ERα and/or ERβ in the hippocampus of Swiss female mice. First, we showed that immediately post-training intraperitoneal (i.p.) injection of E2 (0.2mg/kg) allowed object recognition memory to persist 48h in ovariectomized (OVX) Swiss female mice. This result indicates that Swiss female mice are sensitive to the promnesic effects of E2 and is in accordance with other studies, which used C57/BL6 female mice. To verify if the activation of hippocampal ERα or ERβ would be sufficient to improve object memory, we used PPT and DPN, which are selective ERα and ERβ agonists, respectively. We found that PPT, but not DPN, improved object memory in Swiss female mice. However, DPN was able to improve memory in C57/BL6 female mice, which is in accordance with other studies. Next, we tested if the E2 effect on improving object memory depends on ER activation in the hippocampus. Thus, we tested if the infusion of intra-hippocampal TPBM and PHTPP, selective antagonists of ERα and ERβ, respectively, would block the memory enhancement effect of E2. Our results showed that TPBM, but not PHTPP, blunted the promnesic effect of E2, strongly suggesting that in Swiss female mice, the ERα and not the ERβ is the receptor involved in the promnesic effect of E2. It was already demonstrated that E2, as well as PPT and DPN, increase the phospho-ERK2 level in the dorsal hippocampus of C57/BL6 mice. Here we observed that PPT increased phospho-ERK1, while DPN decreased phospho-ERK2 in the dorsal hippocampus of Swiss female mice subjected to the object recognition sample phase. Taken together, our results suggest that the type of receptor as well as the molecular mechanism used by E2 to improve object memory may differ in Swiss female mice.

Estrogen increases expression of aquaporin‐2 protein in the inner medullary collecting duct (1137.3)

Previous studies of sex differences in renal water handling have focused on vasopressin and the V2 receptor rather than possible differences in their downstream target, aquaporin‐2 (AQP2). We report that inner medullary glycosylated and non‐glycosylated AQP2 protein levels are approximately 30‐40% lower in male (0.69±0.04 and 0.62±0.07 arbitrary units, a.u., n=5) compared to female C57BL/6 mice (1.00±0.08 and 1.00±0.16 a.u., n=4; P<0.05). Immunofluorescent staining and Western blot analysis revealed that freshly isolated collecting duct cells and mpk‐CCD‐clone II cells express estrogen receptor (ER) β, the ER splice variants ERα36 and ERα46, but little if any full‐length ERα66. Exposure of mpk‐CCD‐clone II cells to 10 nM estrogen for 24 h significantly increased AQP2 protein expression (2.6±0.4 vs 0.9±0.2 a.u. for vehicle, n=3; P<0.05), whereas exposure to the selective ERβ agonist DPN at 10 nM had no significant effect. Inner medullary AQP2 mRNA levels were not significantly different between male and female C57BL/6 mice (2‐ΔΔCT = 1.03±0.12 and 1.09±0.08, n=6 per group; P>0.05). Thus our data show that estrogen increases collecting duct AQP2 protein levels, potentially via a mechanism independent of gene transcription.

Inhibition of lymphoma vascularization and dissemination by estrogen receptor β agonists

AbstractMost lymphomas show an increased incidence and poorer prognosis in males vs females, suggesting endocrine regulation. We have previously shown that tumor growth in vivo of a murine T-cell–derived lymphoma is repressed following activation of estrogen receptor β (ERβ, ESR2). By using ERβ-deficient mice, we now demonstrate that this inhibition is mediated via a direct effect on the tumor cells and not on the microenvironment. Furthermore, we show that the growth-suppressing effects of ERβ agonist are also valid for human B-cell lymphomas as demonstrated in tumors derived from Granta-519 mantle cell lymphoma (MCL) and Raji Burkitt lymphoma (BL) cells. In Granta-519 MCL tumors, activation of ERβ reduced expression of BAFF and GRB7, 2 important molecules involved in B-cell proliferation and survival. Importantly, activation of ERβ inhibited angiogenesis and lymphangiogenesis, possibly mediated by impaired vascular endothelial growth factor C expression. Furthermore, using disseminating Raji BL cells, we show that ERβ activation reduces dissemination of grafted Raji BL tumors. We also show by immunohistochemistry that ERβ is expressed in primary MCL tissue. These results suggest that targeting ERβ with agonists may be valuable in the treatment of some lymphomas, affecting several aspects of the malignant process, including proliferation, vascularization, and dissemination.

Activation of a Gq-coupled membrane estrogen receptor rapidly attenuates α2-adrenoceptor-induced antinociception via an ERK I/II-dependent, non-genomic mechanism in the female rat

Though sex differences in pain and analgesia are known, underlying mechanisms remain elusive. This study addresses the selective contribution of membrane estrogen receptors (mERs) and mER-initiated non-genomic signaling mechanisms in our previously reported estrogen-induced attenuation of α2-adrenoceptor-mediated antinociception. By selectively targeting spinal mERs in ovariectomized female rats using β-estradiol 6-(O-carboxy-methyl)oxime bovine serum albumin (E2BSA) (membrane impermeant estradiol analog), and ERα selective agonist 4,4′,4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT), ERβ selective agonist 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN), G-protein-coupled estrogen receptor 30 (GPR30) agonist G1 and Gq-coupled mER (Gq-mER) agonist STX, we provide strong evidence that Gq-mER activation may solely contribute to suppressing clonidine (an α2-adrenoceptor agonist)-induced antinociception, using the nociceptive tail-flick test. Increased tail-flick latencies (TFLs) by intrathecal (i.t.) clonidine were not significantly altered by i.t. PPT, DPN, or G1. In contrast, E2BSA or STX rapidly and dose-dependently attenuated clonidine-induced increase in TFL. ICI 182,780, the ER antagonist, blocked this effect. Consistent with findings with the lack of effect of ERα and ERβ agonists that modulate receptor-regulated transcription, inhibition of de novo protein synthesis using anisomycin also failed to alter the effect of E2BSA or STX, arguing against a contribution of genomic mechanisms. Immunoblotting of spinal tissue revealed that mER activation increased levels of phosphorylated extracellular signal-regulated kinase (ERK) but not of protein kinase A (PKA) or C (PKC). In vivo inhibition of ERK with U0126 blocked the effect of STX and restored clonidine antinociception. Although estrogen-induced delayed genomic mechanisms may still exist, data presented here indicate that Gq-mER may solely mediate estradiol-induced attenuation of clonidine antinociception via a rapid, reversible, and ERK-dependent, non-genomic mechanism, suggesting that Gq-mER blockade might provide improved analgesia in females.

Ovariectomy and Subsequent Treatment with Estrogen Receptor Agonists Tune the Innate Immune System of the Hippocampus in Middle-Aged Female Rats

The innate immune system including microglia has a major contribution to maintenance of the physiological functions of the hippocampus by permanent monitoring of the neural milieu and elimination of tissue-damaging threats. The hippocampus is vulnerable to age-related changes ranging from gene expression to network connectivity. The risk of hippocampal deterioration increases with the decline of gonadal hormone supply. To explore the impact of hormone milieu on the function of the innate immune system in middle-aged female rats, we compared mRNA expression in the hippocampus after gonadal hormone withdrawal, with or without subsequent estrogen replacement using estradiol and isotype-selective estrogen receptor (ER) agonists. Targeted profiling assessed the status of the innate immune system (macrophage-associated receptors, complement, inhibitory neuronal ligands), local estradiol synthesis (P450 aromatase) and estrogen reception (ER). Results established upregulation of macrophage-associated (Cd45, Iba1, Cd68, Cd11b, Cd18, Fcgr1a, Fcgr2b) and complement (C3, factor B, properdin) genes in response to ovariectomy. Ovariectomy upregulated Cd22 and downregulated semaphorin3A (Sema3a) expression, indicating altered neuronal regulation of microglia. Ovariectomy also led to downregulation of aromatase and upregulation of ERα gene. Of note, analogous changes were observed in the hippocampus of postmenopausal women. In ovariectomized rats, estradiol replacement attenuated Iba1, Cd11b, Fcgr1a, C3, increased mannose receptor Mrc1, Cd163 and reversed Sema3a expression. In contrast, reduced expression of aromatase was not reversed by estradiol. While the effects of ERα agonist closely resembled those of estradiol, ERβ agonist was also capable of attenuating the expression of several macrophage-associated and complement genes. These data together indicate that the innate immune system of the aging hippocampus is highly responsive to the gonadal hormone milieu. In ovariectomized female rats, estradiol replacement exerts potent immunomodulatory effects including attenuation of microglia sensitization, initiation of M2-like activation and modulation of complement expression by targeting hippocampal neurons and glial cells through ERα and ERβ.

Erb-041, an estrogen receptor-β agonist, inhibits skin photocarcinogenesis in SKH-1 hairless mice by downregulating the WNT signaling pathway.

Estrogen receptors (ER), including ER-α and ER-β, are known to regulate multiple biologic responses in various cell types. The expression of ER-β is lost in various cancers. ER-β agonists were shown to modulate inflammation, cancer cell proliferation, and differentiation. Here, we investigated the cancer chemopreventive properties of Erb-041, an ER-β agonist, using a model of UVB-induced photocarcinogenesis in SKH-1 mice. Erb-041 significantly reduced UVB-induced carcinogenesis. Tumor numbers and volume were reduced by 60% and 84%, respectively, in the Erb-041-treated group as compared with UVB (alone) control. This inhibition in tumorigenesis was accompanied by the decrease in proliferating cell nuclear antigen (PCNA), cyclin D1, VEGF, and CD31, and an increase in apoptosis. The lost ER-β expression in squamous cell carcinomas (SCC) was significantly recovered by Erb-041 treatment. In addition, the UVB-induced inflammatory responses were remarkably reduced. Myeloperoxidase activity, levels of cytokines (interleukin (IL)-1β, IL-6, and IL-10), and expression of p-ERK (extracellular signal-regulated kinase) 1/2, p-p38, p-IκB, iNOS, COX-2, and nuclear NF-κBp65 were diminished. The number of tumor-associated inflammatory cells (GR-1(+)/CD11b(+) and F4/80(+)) was also decreased. Tumors excised from Erb-041-treated animal were less invasive and showed reduced epithelial-mesenchymal transition (EMT). The enhanced expression of E-cadherin with the concomitantly reduced expression of N-cadherin, Snail, Slug, and Twist characterized these lesions. The WNT/β-catenin signaling pathway, which underlies pathogenesis of skin cancer, was found to be downregulated by Erb-041 treatment. Similar but not identical changes in proliferation and EMT regulatory proteins were noticed following treatment of tumor cells with a WNT signaling inhibitor XAV939. Our results show that Erb-041 is a potent skin cancer chemopreventive agent that acts by dampening the WNT/β-catenin signaling pathway.

Targeting Estrogen Receptor-β for the Prevention of Nonmelanoma Skin Cancer

The potential for targeting estrogen receptor (ER)-β in various cancer models has been gaining considerable attention in recent years. In this issue of the journal, Chaudhary and colleagues demonstrate markedly decreased ultraviolet B (UVB)-induced skin cancer in a mouse model using a highly specific ER-β agonist, ERB-041. The mechanisms that underlie this strong inhibitory effect are mediated by inhibition of proinflammatory signaling and epithelial-mesenchymal transition (EMT). The changes in EMT were due in part to modulation of WNT/β-catenin signaling. Collectively, the results from these studies provide important new insights into the mechanisms by which the ER-β agonist ERB-041 inhibits UVB-induced skin cancer and opens the door for future studies that could examine combinatorial approaches for UVB-dependent skin cancer chemoprevention.

Effects of Neuron-Specific Estrogen Receptor (ER) α and ERβ Deletion on the Acute Estrogen Negative Feedback Mechanism in Adult Female Mice

The negative feedback mechanism through which 17β-estradiol (E2) acts to suppress the activity of the GnRH neurons remains unclear. Using inducible and cell-specific genetic mouse models, we examined the estrogen receptor (ER) isoforms expressed by neurons that mediate acute estrogen negative feedback. Adult female mutant mice in which ERα was deleted from all neurons in the neonatal period failed to exhibit estrous cycles or negative feedback. Adult mutant female mice with neonatal neuronal ERβ deletion exhibited normal estrous cycles, but a failure of E2 to suppress LH secretion was seen in ovariectomized mice. Mutant mice with a GnRH neuron-selective deletion of ERβ exhibited normal cycles and negative feedback, suggesting no critical role for ERβ in GnRH neurons in acute negative feedback. To examine the adult roles of neurons expressing ERα, an inducible tamoxifen-based Cre-LoxP approach was used to ablate ERα from neurons that express calmodulin kinase IIα in adults. This resulted in mice with no estrous cycles, a normal increase in LH after ovariectomy, but an inability of E2 to suppress LH secretion. Finally, acute administration of ERα- and ERβ-selective agonists to adult ovariectomized wild-type mice revealed that activation of ERα suppressed LH secretion, whereas ERβ agonists had no effect. This study highlights the differences in adult reproductive phenotypes that result from neonatal vs adult ablation of ERα in the brain. Together, these experiments expand previous global knockout studies by demonstrating that neurons expressing ERα are essential and probably sufficient for the acute estrogen negative feedback mechanism in female mice.

Psoralidin, a coumestan analogue, as a novel potent estrogen receptor signaling molecule isolated from Psoralea corylifolia

A novel biological activity of psoralidin as an agonist for both estrogen receptor (ER)α and ERβ agonist has been demonstrated in our study. Psoralidin has been characterized as a full ER agonist, which activates the classical ER-signaling pathway in both ER-positive human breast and endometrial cell lines as well as non-human cultured cells transiently expressing either ERα or ERβ. The estrogenic activity was determined using the relative expression levels of either reporter or the endogenous genes dependent on the agonist-bound ER to the estrogen response element (ERE). Psoralidin at 10μM was able to induce the maximum reporter gene expression corresponding to that of E2-treated cells and such activation of the ERE-reporter gene by psoralidin was completely abolished by the cotreatment of a pure ER antagonist, implying that the biological activities of psoralidin are mediated by ER. Psoralidin was also able to induce the endogenous estrogen-responsive gene, pS2, in human breast cancer cells MCF-7. It was observed that activation of the classical ER-signaling pathway by psoralidin is mediated via induction of ER conformation by psoralidin and direct binding of the psoralidin–ER complex to the EREs present in the promoter region of estrogen-responsive genes, as shown by chromatin immunoprecipitation assay results. Finally, molecular docking of psoralidin to the ligand binding pocket of the ERα showed that psoralidin is able to mimic the binding interactions of E2, and thus, it could act as an ER agonist in the cellular environment.

Signaling mechanisms involved in the acute effects of estradiol on 5-HT clearance.

Estradiol was found previously to have an antidepressant-like effect and to block the ability of selective serotonin reuptake inhibitors (SSRIs) to have an antidepressant-like effect. The antidepressant-like effect of estradiol was due to estrogen receptor β (ERβ) and/or GPR30 activation, whereas estradiol's blockade of the effect of an SSRI was mediated by ERα. This study focuses on investigating signaling pathways as well as interacting receptors associated with these two effects of estradiol. In vivo chronoamperometry was used to measure serotonin transporter (SERT) function. The effect of local application of estradiol or selective agonists for ERα (PPT) or ERβ (DPN) into the CA3 region of the hippocampus of ovariectomized (OVX) rats on 5-hydroxytryptamine (5-HT) clearance as well as on the ability of fluvoxamine to slow 5-HT clearance was examined after selective blockade of signaling pathways or that of interacting receptors. Estradiol- or DPN-induced slowing of 5-HT clearance mediated by ERβ was blocked after inhibition of MAPK/ERK1/2 but not of PI3K/Akt signaling pathways. This effect also involved interactions with TrkB, and IGF-1 receptors. Estradiol's or PPT's inhibition of the fluvoxamine-induced slowing of 5-HT clearance mediated by ERα, was blocked after inhibition of either MAPK/ERK1/2 or PI3K/Akt signaling pathways. This effect involved interactions with the IGF-1 receptor and with the metabotropic glutamate receptor 1, but not with TrkB. This study illustrates some of the signaling pathways required for the effects of estradiol on SERT function, and particularly shows that ER subtypes elicit different as well as common signaling pathways for their actions.

17β-estradiol promotes cholesterol efflux from vascular smooth muscle cells through a liver X receptor α-dependent pathway

Estrogen has pleiotropic effects on the cardiovascular diseases, yet the underlying mechanisms remain incompletely understood. Cholesterol efflux is a key mechanism through which to prevent foam cell formation and the development of atherosclerosis. Recent studies highlight the role of vascular smooth muscle cell (VSMC)-derived foam cells in atherogenesis. However, it remains unclear whether estrogen promotes cholesterol efflux from VSMCs and inhibits VSMC-derived foam cell formation. In the present study, we demonstrated that 17β-estradiol (E2) markedly enhanced cholesterol efflux to apolipoprotein (apo)A-1 and high-density lipoprotein (HDL) and attenuated oxidized low-density lipoprotein (ox-LDL) induced cholesteryl ester accumulation in VSMCs, which was associated with an increase in the expression of ATP-binding cassette transporters ABCA1 and ABCG1. The upregulation of ABCA1 and ABCG1 expression by E2 resulted from liver X receptor (LXR)α activation, which was confirmed by the prevention of the expression of ABCA1 and ABCG1 after inhibition of LXRα with a pharmacological inhibitor or small interfering RNA (siRNA). Furthermore, E2 increased LXRα, ABCA1 and ABCG1 expression in VSMCs via the estrogen receptor (ER), and the involvement of ERβ was confirmed by the use of selective ERα or ERβ antagonists (MPP and PHTPP) and agonists (PPT and DPN). These findings suggest that E2 promotes cholesterol efflux from VSMCs and reduces VSMC-derived foam cell formation via ERβ- and LXRα-dependent upregulation of ABCA1 and ABCG1 and provide novel insights into the anti-atherogenic properties of estrogen.

Estrogen Regulates Protein Synthesis and Actin Polymerization in Hippocampal Neurons through Different Molecular Mechanisms

Estrogen rapidly modulates hippocampal synaptic plasticity by activating selective membrane-associated receptors. Reorganization of the actin cytoskeleton and stimulation of mammalian target of rapamycin (mTOR)-mediated protein synthesis are two major events required for the consolidation of hippocampal long-term potentiation and memory. Estradiol regulates synaptic plasticity by interacting with both processes, but the underlying molecular mechanisms are not yet fully understood. Here, we used acute rat hippocampal slices to analyze the mechanisms underlying rapid changes in mTOR activity and actin polymerization elicited by estradiol. Estradiol-induced mTOR phosphorylation was preceded by rapid and transient activation of both extracellular signal-regulated kinase (ERK) and protein kinase B (Akt) and by phosphatase and tensin homolog (PTEN) degradation. These effects were prevented by calpain and ERK inhibitors. Estradiol-induced mTOR stimulation did not require activation of classical estrogen receptors (ER), as specific ERα and ERβ agonists (PPT and DPN, respectively) failed to mimic this effect, and ER antagonists could not block it. Estradiol rapidly activated both RhoA and p21-activated kinase (PAK). Furthermore, a specific inhibitor of RhoA kinase (ROCK), H1152, and a potent and specific PAK inhibitor, PF-3758309, blocked estradiol-induced cofilin phosphorylation and actin polymerization. ER antagonists also blocked these effects of estrogen. Consistently, both PPT and DPN stimulated PAK and cofilin phosphorylation as well as actin polymerization. Finally, the effects of estradiol on actin polymerization were insensitive to protein synthesis inhibitors, but its stimulation of mTOR activity was impaired by latrunculin A, a drug that disrupts actin filaments. Taken together, our results indicate that estradiol regulates local protein synthesis and cytoskeletal reorganization via different molecular mechanisms and signaling pathways.