The major etiological factor for cervical cancer is the high-risk human papillomavirus (HPV), which encodes E6 and E7 oncogenes. However, HPV is not sufficient and estrogen has been proposed as an etiological cofactor for the disease. Its requirement has been demonstrated in mouse models for HPV-associated cervical cancer (e.g., K14E7 transgenic mice). Although germline knockout of estrogen receptor alpha (ERα) renders mice resistant to cervical cancer, the cell type-specific requirement for ERα is not known. In this study, we demonstrate that temporal deletion of stromal ERα induced complete regression of cervical dysplasia in K14E7 mice. Our results strongly support the hypothesis that stromal ERα is necessary for HPV-induced cervical carcinogenesis and implicate paracrine mechanisms involving ERα signaling in the development of estrogen-dependent cervical cancers. This is the first evidence to support the importance of stromal ERα in estrogen-dependent neoplastic disease of the female reproductive tract.
cervical cancer; HPV; stromal ERα; transgenic mouse model
Psychostimulant effects are enhanced by ovarian hormones in women and female rodents. Estradiol increases behavioral responses to psychostimulants in women and female rats, although the underlying mechanism is unknown. This study utilized mice to investigate the time frame and receptor mediation of estradiol’s enhancement of cocaine-induced behavior as mice enable parallel use of genetic, surgical and pharmacological methods. The spontaneous behavior of Sham and Ovariectomized (Ovx) female wild-type (WT) mice was determined during habituation to a novel environment and after cocaine administration. Ovx mice were replaced with vehicle (sesame oil) or 17β-estradiol (E2) for 2 days or 30 min prior to a cocaine challenge to investigate the time course of E2’s effects. To examine receptor mediation of estradiol effects, Ovx mice replaced for 2 days with either the ERα-selective agonist PPT or the ERβ-selective agonist DPN were compared to Sham mice, and mice lacking either ERα (αERKO) or ERβ (βERKO) were compared to WT littermates. Ovx mice exhibited fewer ambulations during habituation than Sham females. Cocaine-induced increases in behavioral ratings were greater in Sham than in Ovx mice. Two days but not 30 min of E2 replacement in Ovx mice increased cocaine responses to Sham levels. PPT replacement also increased the cocaine response relative to vehicle- or DPN- treated Ovx mice. αERKO mice displayed modestly attenuated behavioral responses to novelty and cocaine compared to αWT littermates, but no behavioral differences were found between βERKO and βWT mice. These results suggest that E2 enhances cocaine-stimulated locomotion in mice predominantly through ERα.
Behavior; Cocaine; Dopamine; Estradiol; Mice; Ovariectomy
Liver X receptors (LXR) are stimulated by cholesterol-derived oxysterols and serve as transcription factors to regulate gene expression in response to alterations in cholesterol. In the present study, we investigated the role of LXRs in vascular endothelial cells (ECs) and discovered that LXRβ has nonnuclear function and stimulates EC migration by activating endothelial NOS (eNOS). This process is mediated by estrogen receptor-α (ERα). LXR activation promoted the direct binding of LXRβ to the ligand-binding domain of ERα and initiated an extranuclear signaling cascade that requires ERα Ser118 phosphorylation by PI3K/AKT. Further studies revealed that LXRβ and ERα are colocalized and functionally coupled in EC plasma membrane caveolae/lipid rafts. In isolated aortic rings, LXR activation of NOS caused relaxation, while in mice, LXR activation stimulated carotid artery reendothelialization via LXRβ- and ERα-dependent processes. These studies demonstrate that LXRβ has nonnuclear function in EC caveolae/lipid rafts that entails crosstalk with ERα, which promotes NO production and maintains endothelial monolayer integrity in vivo.
Background: Endocrine-disrupting chemicals (EDCs) influence the activity of estrogen receptors (ERs) and alter the function of the endocrine system. However, the diversity of EDC effects and mechanisms of action are poorly understood.
Objectives: We examined the agonistic activity of EDCs through ERα and ERβ. We also investigated the effects of EDCs on ER-mediated target genes.
Methods: HepG2 and HeLa cells were used to determine the agonistic activity of EDCs on ERα and ERβ via the luciferase reporter assay. Ishikawa cells stably expressing ERα were used to determine changes in endogenous ER target gene expression by EDCs.
Results: Twelve EDCs were categorized into three groups on the basis of product class and similarity of chemical structure. As shown by luciferase reporter analysis, the EDCs act as ER agonists in a cell type– and promoter-specific manner. Bisphenol A, bisphenol AF, and 2-2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (group 1) strongly activated ERα estrogen responsive element (ERE)-mediated responses. Daidzein, genistein, kaempferol, and coumestrol (group 2) activated both ERα and ERβ ERE-mediated activities. Endosulfan and kepone (group 3) weakly activated ERα. Only a few EDCs significantly activated the “tethered” mechanism via ERα or ERβ. Results of real-time polymerase chain reaction indicated that bisphenol A and bisphenol AF consistently activated endogenous ER target genes, but the activities of other EDCs on changes of ER target gene expression were compound specific.
Conclusion: Although EDCs with similar chemical structures (in the same group) tended to have comparable ERα and ERβ ERE-mediated activities, similar chemical structure did not correlate with previously reported ligand binding affinities of the EDCs. Using ERα-stable cells, we observed that EDCs differentially induced activity of endogenous ER target genes.
E2; EDCs; ERα; ERβ; ERE; ER target genes. Environ Health Perspect 121:459–466 (2013)
Background: Diarylheptanoid (D3) isolated from the medicinal plant, Curcuma comosa, has estrogenic activity.
Objective: We aimed to elucidate the mechanism(s) of D3 action and compare it with that of 17β-estradiol (E2) using both in vitro and in vivo uterine models.
Methods: We used human uterine (Ishikawa) cells to determine the estrogenic action of D3 on the activation and nuclear translocation of estrogen receptor α (ERα). In addition, we further characterized the uterine response to D3 treatment in vivo.
Results: D3 activated an estrogen responsive element (ERE) luciferase reporter through ERα, and molecular modeling suggested that D3 could be accommodated in the ERα binding pocket. Using modified ERα to assay ligand-dependent nuclear translocation, we observed D3-dependent ERα interaction and translocation. In mouse uteri, early- and late-phase estrogen-regulated gene responses were increased in D3-treated ovariectomized wild-type animals, in a manner similar to that of E2; no response was seen in ERα knockout animals. We observed a divergence in estrogen responses after D3 treatment: D3 induced robust DNA synthesis in uterine epithelial cells, linked to an increase in cell-cycle–related genes; however, no increase in uterine weight was observed 24 hr after treatment. D3 also affected uterine progesterone receptor expression patterns similar to E2. When D3 and E2 were administered together, we observed no additive or antagonistic effects of D3 on E2. Our findings suggest that D3 is a weak estrogenic agonist compound.
Conclusion: D3 is a weakly acting phytoestrogen that mimics the mitogenic responses produced by E2 in an ERα-dependent manner, but it is unable to increase uterine weight or enhance or antagonize the effects of estrogen.
diarylheptanoid; ER-dependent; nuclear translocation; phytoestrogen; uterus
gland formation occurs postnatally in an ovary- and steroid-independent manner in many species, including humans. Uterine glands secrete substances that are essential for embryo survival. Disruption of gland development during the postnatal period prevents gland formation, resulting in infertility. Interestingly, stabilization of beta-catenin (CTNNB1) in the uterine stroma causes a delay in gland formation rather than a complete absence of uterine glands. Thus, to determine if a critical postnatal window for gland development exists in mice, we tested the effects of extending the endocrine environment of pregnancy on uterine gland formation by treating neonatal mice with estradiol, progesterone, or oil for 5 days. One uterine horn was removed before puberty, and the other was collected at maturity. Some mice were also ovariectomized before puberty. The hormone-treated mice exhibited a delay in uterine gland formation. Hormone-treatment increased the abundance of uterine CTNNB1 and estrogen receptor alpha (ESR1) before puberty, indicating possible mechanisms for delayed gland formation. Despite having fewer glands, progesterone-treated mice were fertile, suggesting that a threshold number of glands is required for pregnancy. Mice that were ovariectomized before puberty did not undergo further uterine growth or gland development. Finally, to establish the role of the ovary in postpartum uterine gland regeneration, mice were either ovariectomized or given a sham surgery after parturition, and uteri were evaluated 1 wk later. We found that the ovary is not required for uterine growth or gland development following parturition.
Thus, uterine gland development occurs continuously in mice and requires the ovary after puberty, but not after parturition.
Uterine gland development is a continuous process that occurs postnatally, postpubertally, and postpartum, and that requires the ovary after puberty, but not after parturition.
endometrium; female reproductive tract; uterine gland; uterus
Background: Endocrine-disrupting chemicals (EDCs) are widely found in the environment. Estrogen-like activity is attributed to EDCs, such as bisphenol A (BPA), bisphenol AF (BPAF), and zearalenone (Zea), but mechanisms of action and diversity of effects are poorly understood.
Objectives: We used in vitro models to evaluate the mechanistic actions of BPA, BPAF, and Zea on estrogen receptor (ER) α and ERβ.
Methods: We used three human cell lines (Ishikawa, HeLa, and HepG2) representing three cell types to evaluate the estrogen promoter activity of BPA, BPAF, and Zea on ERα and ERβ. Ishikawa/ERα stable cells were used to determine changes in estrogen response element (ERE)-mediated target gene expression or rapid action-mediated effects.
Results: The three EDCs showed strong estrogenic activity as agonists for ERα in a dose-dependent manner. At lower concentrations, BPA acted as an antagonist for ERα in Ishikawa cells and BPAF acted as an antagonist for ERβ in HeLa cells, whereas Zea was only a partial antagonist for ERα. ERE-mediated activation by BPA and BPAF was via the AF-2 function of ERα, but Zea activated via both the AF-1 and AF-2 functions. Endogenous ERα target genes and rapid signaling via the p44/42 MAPK pathway were activated by BPA, BPAF, and Zea.
Conclusion: BPA and BPAF can function as EDCs by acting as cell type–specific agonists (≥ 10 nM) or antagonists (≤ 10 nM) for ERα and ERβ. Zea had strong estrogenic activity and activated both the AF-1 and AF-2 functions of ERα. In addition, all three compounds induced the rapid action-mediated response for ERα.
BPA; BPAF; ERα; ERα mutants; ERβ; ERE; estrogen; zearalenone
Background: Studies have shown that perinatal exposure to the synthetic estrogen diethylstilbestrol (DES) leads to feminization of the seminal vesicle (SV) in male mice, as illustrated by tissue hyperplasia, ectopic expression of the major estrogen-inducible uterine secretory protein lactoferrin (LF), and reduced expression of SV secretory protein IV (SVS IV).
Objectives: The present study was designed to evaluate the role of the estrogen receptor (ER) in this action by using ER-knockout (ERKO) mice.
Methods: Wild-type (WT), ERα-null (αERKO), and ERβ-null (βERKO) male mice were treated with either vehicle or DES (2 μg/day) on neonatal days 1–5. These mice were divided into two groups: In the first group, intact mice were sacrificed at 10 weeks of age; in the second group, mice were castrated at 10 weeks of age, allowed to recover for 10 days, treated with dihydrotestosterone (DHT) or placebo, and sacrificed 2 weeks later. Body weights and SV weights were recorded, and mRNA expression levels of Ltf (lactoferrin), Svs4, and androgen receptor (Ar) were assessed.
Results: In DES-treated intact mice, SV weights were reduced in WT and βERKO mice but not in αERKO mice. DES-treated WT and βERKO males, but not αERKO males, exhibited ectopic expression of LF in the SV. DES treatment resulted in decreased SVS IV protein and mRNA expression in WT males, but no effect was seen in αERKO mice. In addition, DES-treated βERKO mice exhibited reduced Svs4 mRNA expression but maintained control levels of SVS IV protein. In DES-treated castrated mice, DHT implants restored SV weights to normal levels in αERKO mice but not in WT mice, suggesting full androgen responsiveness in αERKO mice.
Conclusions: These data suggest that DES-induced SV toxicity and feminization are primarily mediated by ERα; however, some aspects of androgen response may require the action of ERβ.
development; endocrine disruptor; reproductive tract
Insulin like growth factor–1 (IGF-1) stimulates increased proliferation and survival of mammary epithelial cells and also promotes mammary tumorigenesis. To study the effects of IGF-1 on the mammary gland in vivo, we used BK5.IGF-1 transgenic (Tg) mice. In these mice, IGF-1 overexpression is controlled by the bovine keratin 5 promoter and recapitulates the paracrine exposure of breast epithelium to stromal IGF-1 that is seen in women. Studies have shown that BK5.IGF-1 Tg mice are more susceptible to mammary tumorigenesis than wild-type littermates. Investigation of the mechanisms underlying increased mammary cancer risk, reported here, revealed that IGF-1 preferentially activated the PI3K/Akt pathway in glands from prepubertal Tg mice, resulting in increased cyclin D1 expression and hyperplasia. However, in glands from postpubertal Tg mice, a pathway switch occurred and activation of the Ras/Raf/MAPK pathway predominated, without increased cyclin D1 expression or proliferation. We further showed that in prepubertal Tg glands, signaling was mediated by formation of an ERα/IRS-1 complex, which activated IRS-1 and directed signaling via the PI3K/Akt pathway. Conversely, in postpubertal Tg glands, reduced ERα expression failed to stimulate formation of the ERα/IRS-1 complex, allowing signaling to proceed via the alternate Ras/Raf/MAPK pathway. These in vivo data demonstrate that changes in ERα expression at different stages of development direct IGF-1 signaling and the resulting tissue responses. As ERα levels are elevated during the prepubertal and postmenopausal stages, these may represent windows of susceptibility during which increased IGF-1 exposure maximally enhances breast cancer risk.
Disruption of the Esr1 gene encoding estrogen receptor alpha (ERα) by insertion of a neomycin resistance gene (neo) into exon 2 (αERKO mice) was shown previously to cause infertility in male mice. While full-length ERα protein was not expressed in αERKO mice, alternative splicing resulted in the low level expression of a truncated form lacking the N-terminus A/B domain and containing the DNA- and ligand-binding domains. Thus, it was unclear whether the reproductive phenotype in αERKO males was due only to the lack of full-length ERα or was affected by the presence of the variant ERα isoform. The present study examined male mice with exon 3 of Esr1 deleted, lacking the DNA-binding domain, and null for ERα (Ex3αERKO). Dilation of some seminiferous tubules was apparent in male Ex3αERKO mice as early as postnatal day 10 and was pronounced in all tubules from day 20 onward. At 6 weeks of age, sperm numbers and sperm motility were lower in Ex3αERKO than in wild type mice and the rete testis and efferent ductules were dilated. Mating studies determined that adult Ex3αERKO males were infertile and failed to produce copulatory plugs. Serum testosterone levels and Hsd17b3 and Cyp17a1 transcript levels were significantly higher, but serum estradiol, progesterone, LH and FSH levels and Cyp19a1 transcript levels were not significantly different from those in WT mice. These results confirm and extend those seen in other studies on male mice with exon 3 of Esr1 deleted. In addition, the reproductive phenotype of male Ex3αERKO mice recapitulated the phenotype of αERKO mice, strongly suggesting that the αERKO male infertility was not due to the presence of the DNA-binding domain in the truncated form of ERα and that full-length ERα is essential for maintenance of male fertility.
testis; epididymis; spermatogenesis; sperm; male fertility; estrogen receptor alpha
The failure of pancreatic β cells to adapt to an increasing demand for insulin is the major mechanism by which patients progress from insulin resistance to type 2 diabetes (T2D) and is thought to be related to dysfunctional lipid homeostasis within those cells. In multiple animal models of diabetes, females demonstrate relative protection from β cell failure. We previously found that the hormone 17β-estradiol (E2) in part mediates this benefit. Here, we show that treating male Zucker diabetic fatty (ZDF) rats with E2 suppressed synthesis and accumulation of fatty acids and glycerolipids in islets and protected against β cell failure. The antilipogenic actions of E2 were recapitulated by pharmacological activation of estrogen receptor α (ERα) or ERβ in a rat β cell line and in cultured ZDF rat, mouse, and human islets. Pancreas-specific null deletion of ERα in mice (PERα–/–) prevented reduction of lipid synthesis by E2 via a direct action in islets, and PERα–/– mice were predisposed to islet lipid accumulation and β cell dysfunction in response to feeding with a high-fat diet. ER activation inhibited β cell lipid synthesis by suppressing the expression (and activity) of fatty acid synthase via a nonclassical pathway dependent on activated Stat3. Accordingly, pancreas-specific deletion of Stat3 in mice curtailed ER-mediated suppression of lipid synthesis. These data suggest that extranuclear ERs may be promising therapeutic targets to prevent β cell failure in T2D.
Epidemiological data suggest that estrogen deficiency in post menopausal women may contribute to the severity of AMD. We discovered that 17β-estradiol (E2) was a crucial regulator of the severity of extracelllular matrix turnover (ECM) dysregulation both in vivo and in vitro. We also found in vitro that the presence of estrogen receptor (ER)β regulates MMP-2 activity. Therefore in an attempt to delineate the role of the ER subtypes, female estrogen receptor knockout (ERKO) mice were fed a high-fat diet, and the eyes were exposed to seven 5-second doses of nonphototoxic levels of blue-green light over 2 weeks. Three months after cessation of blue light treatment, transmission electron microscopy was performed to assess severity of deposits, Bruchs membrane changes, and choriocapillaris endothelial morphology. We found that changes in the trimolecular complex of proMMP-2, MMP-14 and TIMP-2 correlated with increased Bruch’s membrane thickening or sub retinal deposit formation (basal laminar deposits) in ERKOβ mice. In addition RPE isolated from ERKO β mice had an increase in expression of total collagen and a decrease in MMP-2 activity. Finally we found that ERK an intermediate signaling molecule in the MMP pathway was activated in RPE isolated from ERKOβ mice. These data suggest that mice which lack ERβ are more susceptible to in vivo injury associated with environmental light and high fat diet.
Interest and concern regarding potentially estrogenic substances have resulted in development of model systems to evaluate mechanisms of such chemicals. Microarray studies have indicated that estradiol (E2)-stimulated uterine responses can be divided into early and late phases. Comparison of E2 uterine transcript profiles and those of other estrogenic chemicals of interest in vivo indicates mechanisms and activities of test compounds.
We compared transcript responses and mechanisms of response using mouse reproductive tracts after treatment with E2, estriol (E3), bisphenol A (BPA), and 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE).
Uterine RNA from ovariectomized wild-type mice, estrogen receptor α (ERα) knockout (αERKO) mice, and mice expressing a DNA-binding–deficient ERα (KIKO) treated with E2, E3, BPA, or HPTE for 2 or 24 hr was analyzed by microarray. Resulting regulated transcripts were compared by hierarchical clustering and correlation analysis, and response patterns were verified by reverse-transcription real-time polymerase chain reaction (RT-PCR).
Both xenoestrogens, BPA and HPTE, showed profiles highly correlated to that of E2 in the early response phase (2 hr), but the correlation diminished in the later response phase (24 hr), similar to the known weak estrogen E3. Both xenoestrogens also mimicked E2 in samples from KIKO mice, indicating that they are able to utilize the indirect tethering mode of ERα signaling. No response was detected in ERα-null uteri, indicating that ERα mediates the responses.
Our study forms a basis on which patterns of response and molecular mechanisms of potentially estrogenic chemicals can be assessed.
BPA; ERα; estrogen; HPTE; microarray; uterus
We showed that 17β-estradiol (E2) favors pancreatic β-cell survival via the estrogen receptor-α (ERα) in mice. E2 activates nuclear estrogen receptors via an estrogen response element (ERE). E2 also activates nongenomic signals via an extranuclear form of ERα and the G protein–coupled estrogen receptor (GPER). We studied the contribution of estrogen receptors to islet survival.
RESEARCH DESIGN AND METHODS
We used mice and islets deficient in estrogen receptor-α (αERKO−/−), estrogen receptor-β (βERKO−/−), estrogen receptor-α and estrogen receptor-β (αβERKO−/−), and GPER (GPERKO−/−); a mouse lacking ERα binding to the ERE; and human islets. These mice and islets were studied in combination with receptor-specific pharmacological probes.
We show that ERα protection of islet survival is ERE independent and that E2 favors islet survival through extranuclear and membrane estrogen receptor signaling. We show that ERβ plays a minor cytoprotective role compared to ERα. Accordingly, βERKO−/− mice are mildly predisposed to streptozotocin-induced islet apoptosis. However, combined elimination of ERα and ERβ in mice does not synergize to provoke islet apoptosis. In αβERKO−/− mice and their islets, E2 partially prevents apoptosis suggesting that an alternative pathway compensates for ERα/ERβ deficiency. We find that E2 protection of islet survival is reproduced by a membrane-impermeant E2 formulation and a selective GPER agonist. Accordingly, GPERKO−/− mice are susceptible to streptozotocin-induced insulin deficiency.
E2 protects β-cell survival through ERα and ERβ via ERE-independent, extra-nuclear mechanisms, as well as GPER-dependent mechanisms. The present study adds a novel dimension to estrogen biology in β-cells and identifies GPER as a target to protect islet survival.
Estrogen receptors (ERs) [ERα (Esr1) and ERβ (Esr2)] are expressed in the human colon, but during the multistep process of colorectal carcinogenesis, expression of both ERα and ERβ is lost, suggesting that loss of ER function might promote colorectal carcinogenesis. Through crosses between an ERα knockout and ApcMin mouse strains, we demonstrate that ERα deficiency is associated with a significant increase in intestinal tumor multiplicity, size and burden in ApcMin/+ mice. Within the normal intestinal epithelium of ApcMin/+ mice, ERα deficiency is associated with an accumulation of nuclear β-catenin, an indicator of activation of the Wnt–β-catenin-signaling pathway, which is known to play a critical role in intestinal cancers. Consistent with the hypothesis that ERα deficiency is associated with activation of Wnt–β-catenin signaling, ERα deficiency in the intestinal epithelium of ApcMin/+ mice also correlated with increased expression of Wnt–β-catenin target genes. Through crosses between an ERβ knockout and ApcMin mouse strains, we observed some evidence that ERβ deficiency is associated with an increased incidence of colon tumors in ApcMin/+ mice. This effect of ERβ deficiency does not involve modulation of Wnt–β-catenin signaling. Our studies suggest that ERα and ERβ signaling modulate colorectal carcinogenesis, and ERα does so, at least in part, by regulating the activity of the Wnt–β-catenin pathway.
Both ovarian and pituitary hormones are required for the pubertal development of the mouse mammary gland. Estradiol directs ductal elongation and branching, while progesterone leads to tertiary branching and alveolar development. The purpose of this investigation was to identify estrogen-responsive genes associated with pubertal ductal growth in the mouse mammary gland in the absence of other ovarian hormones and at different stages of development. We hypothesized that the estrogen-induced genes and their associated functions at early stages of ductal elongation would be distinct from those induced after significant ductal elongation had occurred. Therefore, ovariectomized prepubertal mice were exposed to 17β-estradiol from two to twenty-eight days, and mammary gland global gene expression analyzed by microarray analysis at various times during this period. We found that: a) gene expression changes in our estrogen-only model mimic those changes that occur in normal pubertal development in intact mice, and b) both distinct and overlapping gene profiles were observed at varying extents of ductal elongation, and c) cell proliferation, the immune response, and metabolism/catabolism were the most common functional categories associated with mammary ductal growth. Particularly striking was the novel observation that genes active during carbohydrate metabolism were rapidly and robustly decreased in response to estradiol. Lastly, we identified mammary estradiol-responsive genes that are also co-expressed with Estrogen Receptor α in human breast cancer. In conclusion, our genomic data support the physiological observation that estradiol is one of the primary hormonal signals driving ductal elongation during pubertal mammary development.
microarray; genomic; mouse; estrogen receptor
Steroid hormone receptors function classically in the nucleus as transcription factors. However, recent data indicate that there are also non-nuclear subpopulations of steroid hormone receptors, including estrogen receptors (ERs), that mediate membrane-initiated signaling of unclear basis and significance. Here we have shown that an estrogen-dendrimer conjugate (EDC) that is excluded from the nucleus stimulates endothelial cell proliferation and migration via ERα, direct ERα-Gαi interaction, and endothelial NOS (eNOS) activation. Analysis of mice carrying an estrogen response element luciferase reporter, ER-regulated genes in the mouse uterus, and eNOS enzyme activation further indicated that EDC specifically targets non-nuclear processes in vivo. In mice, estradiol and EDC equally stimulated carotid artery reendothelialization in an ERα- and G protein–dependent manner, and both agents attenuated the development of neointimal hyperplasia following endothelial injury. In contrast, endometrial carcinoma cell growth in vitro and uterine enlargement and MCF-7 cell breast cancer xenograft growth in vivo were stimulated by estradiol but not EDC. Thus, EDC is a non-nuclear selective ER modulator (SERM) in vivo, and in mice, non-nuclear ER signaling promotes cardiovascular protection. These processes potentially could be harnessed to provide vascular benefit without increasing the risk of uterine or breast cancer.
It has been shown that long-term estrogen treatment in gonadectomized female mice increases anxiety levels. On the other hand, a recent study has reported that estrogen may down-regulate the levels of anxiety by acting through estrogen receptor (ER) β. In the present study, we investigated the role of ER-β in the regulation of anxiety levels in female mice after long-term estrogen treatment. Gonadectomized ER-β knockout (βERKO) female mice and their wild type (βWT) littermates were implanted several different doses (experiment 1: 2.0μg/day, experiment 2: 1.0, 0.4, 0.2 or 0.1μg/day) of an estradiol benzoate (EB) or placebo pellet. Ten days after pellet implant, behavioral tests commenced to measure the anxiety levels (experiment 1: light-dark transition test (LDT), experiment 2: LDT, elevated plus maze test (EPM) and social investigation test (SIT)). We found that, at higher-doses, long-term treatment of EB had anxiogenic effects in both β WT and βERKO mice as indicated by a decrease of the time spent in the light side and the number of transitions between two sides during LDT. In contrast, several behavioral measurements indicated that the lower-doses treatment of EB might reduce the anxiety levels possibly through ER-β. Particularly, the anxiolytic effects of EB in the SIT were more pronounced in βWT mice than βERKO mice. Together, the findings in the present study suggest that estrogen may have both anxiolytic and anxiogenic effects in female mice, and that ER-β gene disruption did not affect anxiogenic regulation by estrogen in female mice, but partially affected anxiolytic regulation.
fear; emotion; social anxiety; behavioral tests; knockout mouse
The majority of human cervical cancers are associated with the high-risk human papillomaviruses (HPVs), which encode the potent E6 and E7 oncogenes. Upon prolonged treatment with physiological levels of exogenous estrogen, K14E7 transgenic mice expressing HPV-16 E7 oncoprotein in their squamous epithelia succumb to uterine cervical cancer. Furthermore, prolonged withdrawal of exogenous estrogen results in complete or partial regression of tumors in this mouse model. In the current study we investigated whether estrogen receptor alpha (ERα) is required for the development of cervical cancer in K14E7 transgenic mice. We demonstrate that exogenous estrogen fails to promote either dysplasia or cervical cancer in K14E7/ERα−/− mice despite the continued presence of the presumed cervical cancer precursor cell type, reserve cells, and evidence for E7 expression therein. We also observed that cervical cancers in our mouse models are strictly associated with atypical squamous metaplasia (ASM), which is believed to be the precursor for cervical cancer in women. Consistently, E7 and exogenous estrogen failed to promote ASM in the absence of ERα. We conclude that ERα plays a crucial role at an early stage of cervical carcinogenesis in this mouse model.
cervical cancer; HPV; E7; ERα; transgenic mouse model
Platelets derived from aged (reproductively senescent) female mice with genetic deletion of estrogen receptor beta (βER) are more thrombogenic than those from age-matched wild-type (WT) mice. Intracellular processes contributing to this increased thrombogenicity are not known. Experiments were designed to identify subcellular localization of estrogen receptors and evaluate both glycolytic and mitochondrial energetic processes which might affect platelet activation. Platelets and blood from aged (22–24 months) WT and estrogen receptor β knockout (βERKO) female mice were used in this study. Body, spleen weight, and serum concentrations of follicle-stimulating hormone and 17β-estradiol were comparable between WT and βERKO mice. Number of spontaneous deaths was greater in the βERKO colony (50% compared to 30% in WT) over the course of 24 months. In resting (nonactivated) platelets, estrogen receptors did not appear to colocalize with mitochondria by immunostaining. Lactate production and mitochondrial membrane potential of intact platelets were similar in both groups of mice. However, activities of NADH dehydrogenase, cytochrome bc1 complex, and cytochrome c oxidase of the electron transport chain were reduced in mitochondria isolated from platelets from βERKO compared to WT mice. There were a significantly higher number of phosphatidylserine-expressing platelet-derived microvesicles in the plasma and a greater thrombin-generating capacity in βERKO compared to WT mice. These results suggest that deficiencies in βER affect energy metabolism of platelets resulting in greater production of circulating thrombogenic microvesicles and could potentially explain increased predisposition to thromboembolism in some elderly females.
Aging; Estrogen receptors; Microparticles; Mitochondria; Platelet energy metabolism; Procoagulant activity
Ethylene glycol monomethyl ether (EGME) exposure is associated with impaired reproductive function. The primary metabolite of EGME is methoxyacetic acid (MAA), a short-chain fatty acid that inhibits histone deacetylase activity and alters gene expression.
Because estrogen signaling is necessary for normal reproductive function and modulates gene expression, the estrogen-signaling pathway is a likely target for MAA; however, little is known about the effects of MAA in this regard.
We evaluated the mechanistic effects of MAA on estrogen receptor (ER) expression and estrogen signaling using in vitro and in vivo model systems.
MAA potentiates 17β-estradiol (E2) stimulation of an estrogen-responsive reporter plasmid in HeLa cells transiently transfected with either a human ERα or ERβ expression vector containing a cytomegalovirus (CMV) promoter. This result is attributed to increased exogenous ER expression due to MAA-mediated activation of the CMV promoter. In contrast to its effects on exogenous ER, MAA decreases endogenous ERα expression and attenuates E2-stimulated endogenous gene expression in both MCF-7 cells and the mouse uterus.
These results illustrate the importance of careful experimental design and analysis when assessing the potential endocrine-disrupting properties of a compound to ensure biological responses are in concordance with in vitro analyses. Given the established role of the ER in normal reproductive function, the effects of MAA on the endogenous ER reported here are consistent with the reproductive abnormalities observed after EGME exposure and suggest that these toxicities may be due, at least in part, to attenuation of endogenous ER-mediated signaling.
estrogen receptor; estrogen; methoxyacetic acid; short-chain fatty acid
While premature suture fusion, or craniosynostosis, is a relatively common condition, the cause is often unknown. Estrogens are associated with growth plate fusion of endochondral bones. In the following study, we explore the previously unknown significance of estrogen/estrogen receptor signaling in cranial suture biology.
Firstly, estrogen receptor (ER) expression was examined in physiologically fusing (posterofrontal) and patent (sagittal) mouse cranial sutures by quantitative RT-PCR. Next, the cranial suture phenotype of ER alpha and ER beta knockout (αERKO, βERKO) mice was studied. Subsequently, mouse suture-derived mesenchymal cells (SMCs) were isolated; the effects of 17-β estradiol or the estrogen antagonist Fulvestrant on gene expression, osteogenic and chondrogenic differentiation were examined in vitro. Finally, in vivo experiments were performed in which Fulvestrant was administered subcutaneously to the mouse calvaria. Results showed that increased ERα but not ERβ transcript abundance temporally coincided with posterofrontal suture fusion. The αERKO but not βERKO mouse exhibited delayed posterofrontal suture fusion. In vitro, addition of 17-β estradiol enhanced both osteogenic and chondrogenic differentiation in suture-derived mesenchymal cells, effects reversible by Fulvestrant. Finally, in vivo application of Fulvestrant significantly diminished calvarial osteogenesis, inhibiting suture fusion.
Estrogen signaling through ERα but not ERβ is associated with and necessary for normal mouse posterofrontal suture fusion. In vitro studies suggest that estrogens may play a role in osteoblast and/or chondrocyte differentiation within the cranial suture complex.
Diarylheptanoids isolated from Curcuma comosa Roxb. have been recently identified as phyto estrogens. However, the mechanism underlying their actions has not yet been identified.
We characterized the estrogenic activity of three active naturally occurring diarylheptanoids both in vitro and in vivo.
We characterized mechanisms of estrogenic action of the diarylheptanoids (3S)-1,7-diphenyl-(6E)-6-hepten-3-ol (D1), 1,7-diphenyl-(6E)-6-hepten-3-one (D2), and (3R)-1,7-diphenyl-(4E,6E)-4,6-heptadien-3-ol (D3) by using a real-time polymerase chain reaction assay, a mammalian transfection model, and a uterotrophic assay in mice.
All diarylheptanoids up-regulated estrogen-responsive genes in estrogen-responsive breast cancer cells (MCF-7). In HepG2 cells transfected with estrogen receptor (ER) β or different ERα functional receptor mutants and the Vit-ERE-TATA-Luc reporter gene, all diarylheptanoids induced transcription through a ligand-dependent human ERα-ERE–driven pathway, which was abolished with ICI 182,780 (ER antagonist), whereas only D2 was active with ERβ. An ERα mutant lacking the functional AF2 (activation function 2) region was not responsive to 17β-estradiol (E2) or to any of the diarylheptanoids, whereas ERα lacking the AF1 domain exhibited wild-type–like activity. D3 markedly increased uterine weight and proliferation of the uterine epithelium in ovariectomized mice, whereas D1 and D2 were inactive. D3, like E2, up-regulated lactoferrin (Ltf) gene expression. The responses to D3 in the uterus were inhibited by ICI 182,780. In addition, D3 stimulated both classical (Aqp5) and nonclassical (Cdkn1a) ER-mediated gene regulation.
The results suggest that the D3 diarylheptanoid is an agonist for ER both in vitro and in vivo, and its biological action is ERα selective, specifically requiring AF2 function, and involves direct binding via ER as well as ERE-independent gene regulation.
Curcuma comosa; diarylheptanoid; ER dependent; ERE dependent; ERE independent; estrogen-regulated genes; uterotrophic activity
The roles of sex hormones as modulators of lung function and disease have received significant attention as differential sex responses to various lung insults have been recently reported. The present study used a bleomycin-induced pulmonary fibrosis model in C57BL/6 mice to examine potential sex differences in physiological and pathological outcomes. Endpoints measured included invasive lung function assessment, immunological response, lung collagen deposition, and a quantitative histological analysis of pulmonary fibrosis. Male mice had significantly higher basal static lung compliance than female mice (P < 0.05) and a more pronounced decline in static compliance after bleomycin administration when expressed as overall change or percentage of baseline change (P < 0.05). In contrast, there were no significant differences between the sexes in immune cell infiltration into the lung or in total lung collagen content after bleomycin. Total lung histopathology scores measured using the Ashcroft method did not differ between the sexes, while a quantitative histopathology scoring system designed to determine where within the lung the fibrosis occurred indicated a tendency toward more fibrosis immediately adjacent to airways in bleomycin-treated male versus female mice. Furthermore, castrated male mice exhibited a female-like response to bleomycin while female mice given exogenous androgen exhibited a male-like response. These data indicate that androgens play an exacerbating role in decreased lung function after bleomycin administration, and traditional measures of fibrosis may miss critical differences in lung function between the sexes. Sex differences should be carefully considered when designing and interpreting experimental models of pulmonary fibrosis in mice.
fibrosis; bleomycin; sex; respiratory mechanics
Estrogens are involved in the hypothalamic control of multiple homeostatic functions including reproduction, stress responses, energy metabolism, sleep cycles, temperature regulation, and motivated behaviors. The critical role of 17β-estradiol (E2) is evident in hypoestrogenic states (e.g., postmenopause) in which many of these functions go awry. The actions of E2 in the brain have been attributed to the activation of estrogen receptors α and β through nuclear, cytoplasmic, or membrane actions. However, we have identified a putative membrane-associated estrogen receptor that is coupled to desensitization of GABAB and μ-opioid receptors in guinea pig and mouse hypothalamic proopiomelanocortin neurons. We have synthesized a new nonsteroidal compound, STX, which selectively targets the Gα q-coupled phospholipase C–protein kinase C–protein kinase A pathway, and have established that STX is more potent than E2 in mediating this desensitization in an ICI 182, 780-sensitive manner in both guinea pig and mouse neurons. Both E2 and STX were fully efficacious in estrogen receptor α,β knock-out mice. Moreover, in vivo treatment with STX, similar to E2, attenuated the weight gain in hypoestrogenic female guinea pigs. Therefore, this membrane-delimited signaling pathway plays a critical role in the control of energy homeostasis and may provide a novel therapeutic target for treatment of postmenopausal symptoms and eating disorders in females.
GPCR; body weight; GABAB receptor; intracellular signaling; potassium channels; POMC neurons