Katherine A. Burns

Estrogen receptors and human disease: an update

A myriad of physiological processes in mammals are influenced by estrogens and the estrogen receptors (ERs), ERα and ERβ. As we reviewed previously, given the widespread role for estrogen in normal human physiology, it is not surprising that estrogen is implicated in the development or progression of a number of diseases. In this review, we are giving a 5-year update of the literature regarding the influence of estrogens on a number of human cancers (breast, ovarian, colorectal, prostate, and endometrial), endometriosis, fibroids, and cardiovascular disease. A large number of sophisticated experimental studies have provided insights into human disease, but for this review, the literature citations were limited to articles published after our previous review (Deroo and Korach in J Clin Invest 116(3):561–570, 2006) and will focus in most cases on human data and clinical trials. We will describe the influence in which estrogen’s action, through one of or both of the ERs, mediates the aforementioned human disease states.

Differential estrogenic actions of endocrine-disrupting chemicals bisphenol A, bisphenol AF, and zearalenone through estrogen receptor α and β in vitro.

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.

Comprehensive analysis of gene expression in rat and human hepatoma cells exposed to the peroxisome proliferator WY14,643

Peroxisome proliferators (PPs) are an important class of chemicals that act as hepatic tumor promoters in laboratory rodents. The key target for PPs is the nuclear receptor peroxisome proliferator-activated receptor-alpha (PPARalpha) and these chemicals cause cancer by altering the expression of a subset of genes involved in cell growth regulation. The purpose of the present study was to utilize high-density gene expression arrays to examine the genes regulated by the potent PP Wy14,643 (50 microM, 6 h) in both rat (FaO) and human (HepG2) hepatoma cells. Treatment of FaO cells, but not HepG2, revealed the expected fatty acid catabolism genes. However, a larger than expected number of protein kinases, phosphatases, and signaling molecules were also affected exclusively in the FaO cells, including MAPK-phosphatase 1 (MKP-1), Janus-activated kinases 1 and 2 (JAK1 and 2), and glycogen synthetase kinase alpha and beta (GSKalpha and beta). The mRNA accumulation of these genes as well as the protein level for GSK3alpha, JAK1, and JAK2 and MKP-1 activity was corroborated. Due to the importance of MKP-1 in cell signaling, this induction was examined further and was found to be controlled, at least in part, at the level of the genes promoter. Interestingly, overexpression of MKP-1 in turn affected the constitutive activity of PPARalpha. Taken together, the gene expression arrays revealed an important subset of PP-regulated genes to be kinases and phosphatases. These enzymes not only would affect growth factor signaling and cell cycle control but also could represent feedback control mechanisms and modulate the activity of PPARalpha.

Endocrine-Disrupting Chemicals (EDCs): In Vitro Mechanism of Estrogenic Activation and Differential Effects on ER Target Genes

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.

Role of Estrogen Receptor Signaling Required for Endometriosis-Like Lesion Establishment in a Mouse Model

Endometriosis results from ectopic invasion of endometrial tissue within the peritoneal cavity. Aberrant levels of the estrogen receptor (ER), ERα and ERβ, and higher incidence of autoimmune disorders are observed in women with endometriosis. An immunocompetent mouse model of endometriosis was used in which minced uterine tissue from a donor was dispersed into the peritoneal cavity of a recipient. Wild-type (WT), ERα-knockout (αERKO), and βERKO mice were donors or recipients to investigate the roles of ERα, ERβ, and estradiol-mediated signaling on endometriosis-like disease. Mice were treated with vehicle or estradiol, and resulting location, number, and size of endometriosis-like lesions were assessed. In comparison with WT lesions in WT hosts, αERKO lesions in WT hosts were smaller and fewer in number. The effect of ER status and estradiol treatment on nuclear receptor status, proliferation, organization, and inflammation within lesions were examined. αERKO lesions in WT hosts did not form distal to the incision site, respond to estradiol, or proliferate but did have increased inflammation. WT lesions in αERKO hosts did respond to estradiol, proliferate, and show decreased inflammation with treatment, but surprisingly, progesterone receptor expression and localization remained unchanged. Only minor differences were observed between WT lesions in βERKO hosts and βERKO lesions in WT hosts, demonstrating the estradiol-mediated signaling responses are predominately through ERα. In sum, these results suggest ER in both endometriosis-like lesions and their environment influence lesion characteristics, and understanding these interactions may play a critical role in elucidating this enigmatic disease.

Selective Mutations in Estrogen Receptor α D-domain Alters Nuclear Translocation and Non-estrogen Response Element Gene Regulatory Mechanisms

The three main mechanisms of ERα action are: 1) nuclear, genomic, direct DNA binding, 2) nuclear, genomic, “tethered”-mediated, protein-protein interactions, and 3) non-nuclear, non-genomic, rapid action responses. Reports suggest the D-domain or hinge region of ERα plays an important role in mechanisms 1 and 2 above. Studies demonstrating the functionality of the ERα hinge region have resected the full D-domain; therefore, site directed mutations were made to attribute precise sequence functionality to this domain. This study focuses on the characterization and properties of three novel site directed ERα- D-domain mutants. The Hinge 1 (H1) ERα mutant has disrupted nuclear localization, can no longer perform tethered mediated responses and has lost interaction with c-Jun, but retains estrogen response element (ERE)-mediated functions as demonstrated by confocal microscopy, reporter assays, endogenous gene expression and co-immunoprecipitation. The H2 ERα mutant is non-nuclear, but translocates to the nucleus with estradiol (E2) treatment and maintains ERE-mediated functionality. The H2+NES ERα mutant does not maintain nuclear translocation with hormone binding, no longer activates ERE-target genes, functions in ERE- or tethered-mediated luciferase assays, but does retain the non-genomic, non-nuclear, rapid action response. These studies reveal the sequence(s) in the ERα hinge region that are involved in tethered-mediated actions as well as nuclear localization and attribute important functionality to this region of the receptor. In addition, the properties of these ERα mutants will allow future studies to further dissect and characterize the three main ERα mechanisms of action and determine the mechanistic role each action has in estrogen hormone regulation.

Diethylstilbestrol (DES)-Stimulated Hormonal Toxicity is Mediated by ERα Alteration of Target Gene Methylation Patterns and Epigenetic Modifiers (DNMT3A, MBD2, and HDAC2) in the Mouse Seminal Vesicle

Background: Diethylstilbestrol (DES) is a synthetic estrogen associated with adverse effects on reproductive organs. DES-induced toxicity of the mouse seminal vesicle (SV) is mediated by estrogen receptor α (ERα), which alters expression of seminal vesicle secretory protein IV (Svs4) and lactoferrin (Ltf) genes. Objectives: We examined a role for nuclear receptor activity in association with DNA methylation and altered gene expression. Methods: We used the neonatal DES exposure mouse model to examine DNA methylation patterns via bisulfite conversion sequencing in SVs of wild-type (WT) and ERα-knockout (αERKO) mice. Results: The DNA methylation status at four specific CpGs (–160, –237, –306, and –367) in the Svs4 gene promoter changed during mouse development from methylated to unmethylated, and DES prevented this change at 10 weeks of age in WT SV. At two specific CpGs (–449 and –459) of the Ltf gene promoter, DES altered the methylation status from methylated to unmethylated. Alterations in DNA methylation of Svs4 and Ltf were not observed in αERKO SVs, suggesting that changes of methylation status at these CpGs are ERα dependent. The methylation status was associated with the level of gene expression. In addition, gene expression of three epigenetic modifiers—DNMT3A, MBD2, and HDAC2—increased in the SV of DES-exposed WT mice. Conclusion: DES-induced hormonal toxicity resulted from altered gene expression of Svs4 and Ltf associated with changes in DNA methylation that were mediated by ERα. Alterations in gene expression of DNMT3A, MBD2, and HDAC2 in DES-exposed male mice may be involved in mediating the changes in methylation status in the SV. Citation: Li Y, Hamilton KJ, Lai AY, Burns KA, Li L, Wade PA, Korach KS. 2014. Diethylstilbestrol (DES)-stimulated hormonal toxicity is mediated by ERα alteration of target gene methylation patterns and epigenetic modifiers (DNMT3A, MBD2, and HDAC2) in the mouse seminal vesicle. Environ Health Perspect 122:262–268; http://dx.doi.org/10.1289/ehp.1307351

Insufficient Luteinizing Hormone-Induced Intracellular Signaling Disrupts Ovulation in Preovulatory Follicles Lacking Estrogen Receptor-β

Gonadotropin-stimulated estrogen receptor-beta (ERbeta)-null preovulatory follicles exhibit submaximal estradiol production, insufficient acquisition of LH receptor, and attenuated expression of essential ovulatory genes. These observations lead to low ovulatory rates compared with wild-type (WT) follicles. We hypothesize that insufficient LH receptor results in reduced cAMP production after an ovulatory stimulus. Individual preantral follicles were cultured with FSH for 4 d and then induced to ovulate with a single dose of human chorionic gonadotropin (hCG). cAMP levels 1 h after hCG were 50% lower in ERbeta-null than WT follicles. To determine whether the lack of LH receptor, and resulting lack of cAMP, could be bypassed by direct activation of adenylyl cyclase, WT and ERbeta-null follicles were induced to ovulate with forskolin. Ten micromolar forskolin doubled the ovulatory rate of ERbeta-null follicles compared with treatment with hCG ( approximately 50 vs. 25%, respectively). In WT follicles, 10 microm forskolin reduced the ovulation rate compared with hCG (14 vs. 83%, respectively), indicating that high doses of forskolin inhibited WT ovulation. A 10 microm concentration of forskolin induced cAMP levels in ERbeta-null follicles that were comparable to levels produced in WT follicles after hCG and either partially or completely rescued the attenuated expression of LH-responsive genes. These data indicate that direct activation of adenylyl cyclase, resulting in increased production of cAMP, partially rescues the ovulatory response of ERbeta-null follicles, suggesting that insufficient LH receptor and low cAMP levels contribute to their poor ovulatory rates. We also determined that ERbeta-null ovaries exhibit an alteration in the activation of ERK1/2. Our evaluation of the ERbeta-null ovarian phenotype indicates that ERbeta plays a role in facilitating folliculogenesis. We show that expression of ERbeta in preovulatory follicles is required for adequate cAMP production and propose that an optimal level of cAMP is required for hCG-stimulated ovulation.

Biliverdin Reductase A Attenuates Hepatic Steatosis by Inhibition of Glycogen Synthase Kinase (GSK) 3β Phosphorylation of Serine 73 of Peroxisome Proliferator-activated Receptor (PPAR) α

Non-alcoholic fatty liver disease is the most rapidly growing form of liver disease and if left untreated can result in non-alcoholic steatohepatitis, ultimately resulting in liver cirrhosis and failure. Biliverdin reductase A (BVRA) is a multifunctioning protein primarily responsible for the reduction of biliverdin to bilirubin. Also, BVRA functions as a kinase and transcription factor, regulating several cellular functions. We report here that liver BVRA protects against hepatic steatosis by inhibiting glycogen synthase kinase 3β (GSK3β) by enhancing serine 9 phosphorylation, which inhibits its activity. We show that GSK3β phosphorylates serine 73 (Ser(P)73) of the peroxisome proliferator-activated receptor α (PPARα), which in turn increased ubiquitination and protein turnover, as well as decreased activity. Interestingly, liver-specific BVRA KO mice had increased GSK3β activity and Ser(P)73 of PPARα, which resulted in decreased PPARα protein and activity. Furthermore, the liver-specific BVRA KO mice exhibited increased plasma glucose and insulin levels and decreased glycogen storage, which may be due to the manifestation of hepatic steatosis observed in the mice. These findings reveal a novel BVRA-GSKβ-PPARα axis that regulates hepatic lipid metabolism and may provide unique targets for the treatment of non-alcoholic fatty liver disease.

The Naturally Occurring Luteinizing Hormone Surge Is Diminished in Mice Lacking Estrogen Receptor Beta in the Ovary

ABSTRACT Female ESR2-null mice (betaERKO) display defects in ovarian function and are subfertile. Follicular maturation is impaired and explains smaller litters, but betaERKO also produce fewer litters, which may be partially due to inadequate ovulatory signals. To test this, the amplitude and timing of the naturally occurring luteinizing hormone (LH) surge was measured in individual intact betaERKO and wild-type (WT) mice. Vaginal cytology was evaluated daily, and blood samples were taken from mice in proestrus. The amplitude of the LH surge was severely blunted in betaERKO mice compared to WT, but pituitary LH levels revealed no differences. The betaERKO mice did not produce a preovulatory estradiol surge. To determine if the smaller LH surges and the reduced number of litters in betaERKO were due to the lack of ESR2 in the hypothalamic-pituitary axis or due to the absence of ESR2 in the ovary, ovaries were transplanted from WT into betaERKO mice and vice versa. The size of the LH surge was reduced only in mice lacking ESR2 within the ovary, and these mice had fewer litters. Fertility and size of the LH surge were rescued in betaERKO mice receiving a WT ovary. These data provide the first experimental evidence that the LH surge is impaired in betaERKO females and may contribute to their reduced fertility. ESR2 is not necessary within the pituitary and hypothalamus for the generation of a normal LH surge and for normal fertility, but ESR2 is essential within the ovary to provide proper signals.