Estradiol resolves pneumonia via ERß in regulatory T cells.

Current treatments for pneumonia (PNA) are focused on the pathogens. Mortality from PNA-induced acute lung injury (PNA-ALI) remains high, underscoring the need for additional therapeutic targets. Clinical and experimental evidence exists for potential sex differences in PNA survival, with males having higher mortality. In a model of severe pneumococcal PNA, when compared with male mice, age-matched female mice exhibited enhanced resolution characterized by decreased alveolar and lung inflammation and increased numbers of Tregs. Recognizing the critical role of Tregs in lung injury resolution, we evaluated whether improved outcomes in female mice were due to estradiol (E2) effects on Treg biology. E2 promoted a Treg-suppressive phenotype in vitro and resolution of PNA in vivo. Systemic rescue administration of E2 promoted resolution of PNA in male mice independent of lung bacterial clearance. E2 augmented Treg expression of Foxp3, CD25, and GATA3, an effect that required ERß, and not ERα, signaling. Importantly, the in vivo therapeutic effects of E2 were lost in Treg-depleted mice (Foxp3DTR mice). Adoptive transfer of ex vivo E2-treated Tregs rescued Streptococcus pneumoniae-induce PNA-ALI, a salutary effect that required Treg ERß expression. E2/ERß was required for Tregs to control macrophage proinflammatory responses. Our findings support the therapeutic role for E2 in promoting resolution of lung inflammation after PNA via ERß Tregs.

Effects of neural estrogen receptor beta deletion on social and mood-related behaviors and underlying mechanisms in male mice.

Estradiol derived from neural aromatization of testosterone plays a key role in the organization and activation of neural structures underlying male behaviors. This study evaluated the contribution of the estrogen receptor (ER) ß in estradiol-induced modulation of social and mood-related behaviors by using mice lacking the ERß gene in the nervous system. Mutant males exhibited reduced social interaction with same-sex congeners and impaired aggressive behavior. They also displayed increased locomotor activity, and reduced or unaffected anxiety-state level in three paradigms. However, when mice were exposed to unescapable stress in the forced swim and tail suspension tests, they spent more time immobile and a reduced time in swimming and climbing. These behavioral alterations were associated with unaffected circadian and restraint stress-induced corticosterone levels, and unchanged number of tryptophan hydroxylase 2-immunoreactive neurons in the dorsal raphe. By contrast, reduced mRNA levels of oxytocin and arginine-vasopressin were observed in the bed nucleus of stria terminalis, whereas no changes were detected in the hypothalamic paraventricular nucleus. The neural ERß is thus involved to different extent levels in social and mood-related behaviors, with a particular action on oxytocin and arginine-vasopressin signaling pathways of the bed nucleus of stria terminalis, yet the involvement of other brain areas cannot be excluded.

Effects of ERß and ERα on OVX-induced changes in adiposity and insulin resistance.

Loss of ovarian hormones leads to increased adiposity and insulin resistance (IR), increasing the risk for cardiovascular and metabolic diseases. The purpose of this study was to investigate whether the molecular mechanism behind the adverse systemic and adipose tissue-specific metabolic effects of ovariectomy requires loss of signaling through estrogen receptor alpha (ERα) or estrogen receptor ß (ERß). We examined ovariectomized (OVX) and ovary-intactwild-type (WT), ERα-null (αKO), and ERß-null (ßKO) female mice (age ~49 weeks; n = 7-12/group). All mice were fed a phytoestrogen-free diet (<15 mg/kg) and either remained ovary-intact (INT) or were OVX and followed for 12 weeks. Body composition, energy expenditure, glucose tolerance, and adipose tissue gene and protein expression were analyzed. INT αKO were ~25% fatter with reduced energy expenditure compared to age-matched INT WT controls and ßKO mice (all P < 0.001). Following OVX, αKO mice did not increase adiposity or experience a further increase in IR, unlike WT and ßKO, suggesting that loss of signaling through ERα mediates OVX-induced metabolic dysfunction. In fact, OVX in αKO mice (i.e., signaling through ERß in the absence of ERα) resulted in reduced adiposity, adipocyte size, and IR (P < 0.05 for all). ßKO mice responded adversely to OVX in terms of increased adiposity and development of IR. Together, these findings challenge the paradigm that ERα mediates metabolic protection over ERß in all settings. These findings lead us to suggest that, following ovarian hormone loss, ERß may mediate protective metabolic benefits.

Colitis-induced colorectal cancer and intestinal epithelial estrogen receptor beta impact gut microbiota diversity.

Chronic inflammation of the colon (colitis) is a risk factor for colorectal cancer (CRC). Hormone-replacement therapy reduces CRC incidences, and the estrogen receptor beta (ERß/ESR2) has been implicated in this protection. Gut microbiota is altered in both colitis and CRC and may influence the severity of both. Here we test the hypothesis that intestinal ERß impacts the gut microbiota. Mice with and without intestine-specific deletion of ERß (ERßKOVil ) were generated using the Cre-LoxP system. Colitis and CRC were induced with a single intraperitoneal injection of azoxymethane (AOM) followed by administration of three cycles of dextran sulfate sodium (DSS) in drinking water. The microbiota population were characterized by high-throughput 16S rRNA gene sequencing of DNA extracted from fecal samples (N = 39). Differences in the microbiota due to AOM/DSS and absence of ERß were identified through bioinformatic analyses of the 16S-Seq data, and the distribution of bacterial species was corroborated using qPCR. We demonstrate that colitis-induced CRC reduced the gut microbiota diversity and that loss of ERß enhanced this process. Further, the Bacteroidetes genus Prevotellaceae_UCG_001 was overrepresented in AOM/DSS mice compared to untreated controls (3.5-fold, p = 0.004), and this was enhanced in females and in ERßKOVil mice. Overall, AOM/DSS enriched for microbiota impacting immune system diseases and metabolic functions, and lack of ERß in combination with AOM/DSS enriched for microbiota impacting carbohydrate metabolism and cell motility, while reducing those impacting the endocrine system. Our data support that intestinal ERß contributes to a more favorable microbiome that could attenuate CRC development.

Recovery From a Myocardial Infarction Is Impaired in Male C57bl/6 N Mice Acutely Exposed to the Bisphenols and Phthalates That Escape From Medical Devices Used in Cardiac Surgery.

Bisphenols and phthalates leach from medical devices, and this exposure is likely to increase in postcardiac surgery patients. Previous studies suggest that such chemical exposure may impact recovery and wound healing, yet the direct effects of bisphenols and phthalates are unknown in this context. To study the direct effect of clinically based chemical exposures, we measured the metabolites representative of 6 bisphenols and 10 phthalates in men before and after cardiac surgery and then replicated this exposure in a mouse model of cardiac surgery and assessed survival, cardiac function and inflammation. Bisphenol A (BPA), di-ethyl hexyl phthalate (DEHP), butylbenzyl phthalate, di-isodecyl phthalate, and di-n-butyl phthalate metabolites were increased after surgery. DEHP exposure predominated, was positively correlated with duration on the cardiopulmonary bypass machine and exceeded its tolerable daily intake limit by 37-fold. In vivo, C57bl/6 N male mice treated with BPA+phthalates during recovery from surgery-induced myocardial infarction had reduced survival, greater cardiac dilation, reduced cardiac function and increased infiltration of neutrophils, monocytes and macrophages suggesting impaired recovery. Of interest, genetic ablation or estrogen receptor beta (ERß) antagonism did not improve recovery and replacement of DEHP with tri-octyl trimellitate or removal of BPA from the mixture did not ameliorate these effects. To examine the direct effects on inflammation, treatment of human THP-1 macrophages with BPA and phthalates induced a dysfunctional proinflammatory macrophage phenotype with increased expression of M1-type macrophage polarization markers and MMP9 secretion, yet reduced phagocytic activity. These results suggest that chemicals escape from medical devices and may impair patient recovery.

Estrogen receptor beta modulates permeability transition in brain mitochondria.

Recent evidence highlights a role for sex and hormonal status in regulating cellular responses to ischemic brain injury and neurodegeneration. A key pathological event in ischemic brain injury is the opening of a mitochondrial permeability transition pore (MPT) induced by excitotoxic calcium levels, which can trigger irreversible damage to mitochondria accompanied by the release of pro-apoptotic factors. However, sex differences in brain MPT modulation have not yet been explored. Here, we show that mitochondria isolated from female mouse forebrain have a lower calcium threshold for MPT than male mitochondria, and that this sex difference depends on the MPT regulator cyclophilin D (CypD). We also demonstrate that an estrogen receptor beta (ERß) antagonist inhibits MPT and knockout of ERß decreases the sensitivity of mitochondria to the CypD inhibitor, cyclosporine A. These results suggest a functional relationship between ERß and CypD in modulating brain MPT. Moreover, co-immunoprecipitation studies identify several ERß binding partners in mitochondria. Among these, we investigate the mitochondrial ATPase as a putative site of MPT regulation by ERß. We find that previously described interaction between the oligomycin sensitivity-conferring subunit of ATPase (OSCP) and CypD is decreased by ERß knockout, suggesting that ERß modulates MPT by regulating CypD interaction with OSCP. Functionally, in primary neurons and hippocampal slice cultures, modulation of ERß has protective effects against glutamate toxicity and oxygen glucose deprivation, respectively. Taken together, these results reveal a novel pathway of brain MPT regulation by ERß that could contribute to sex differences in ischemic brain injury and neurodegeneration.

Aucubin promotes angiogenesis via estrogen receptor beta in a mouse model of hindlimb ischemia.

Aucubin (AU) is an iridoid glycoside that has been shown to display estrogenic properties and has various pharmacological effects. Herein, we described the angiogenic properties of AU. In the study, hindlimb ischemia was induced by ligation of femoral artery on the right leg of ovariectomized mice. AU treatment significantly accelerated perfusion recovery and reduced tissue injury in mice muscle. Quantification of CD31-positive vessels in hindlimb muscles provided evidences that AU promoted angiogenesis in peripheral ischemia. In addition, results from quantitative PCR and western blot suggested AU induced angiogenesis via vascular endothelial cell growth factor (VEGF)/Akt/endothelial nitric oxide synthase (eNOS) signaling pathway. More interestingly, AU's angiogenic effects could be completely abolished in estrogen receptor beta (ERß) knockout mice. In conclusion, the underlying mechanisms were elucidated that AU produced pro-angiogenic effects through ERß-mediated VEGF signaling pathways. These results expand knowledge about the beneficial effects of AU in angiogenesis and blood flow recovery. It might provide insight into the ERß regulating neovascularisation in hindlimb ischemia and identify AU as a potent new compound used for the treatment of peripheral vascular disease.

ERß-Dependent Direct Suppression of Human and Murine Th17 Cells and Treatment of Established Central Nervous System Autoimmunity by a Neurosteroid.

Multiple sclerosis (MS), an autoimmune disease of the CNS, is mediated by autoreactive Th cells. A previous study showed that the neurosteroid dehydroepiandrosterone (DHEA), when administered preclinically, could suppress progression of relapsing-remitting experimental autoimmune encephalomyelitis (EAE). However, the effects of DHEA on human or murine pathogenic immune cells, such as Th17, were unknown. In addition, effects of this neurosteroid on symptomatic disease, as well as the receptors involved, had not been investigated. In this study, we show that DHEA suppressed peripheral responses from patients with MS and reversed established paralysis and CNS inflammation in four different EAE models, including the 2D2 TCR-transgenic mouse model. DHEA directly inhibited human and murine Th17 cells, inducing IL-10-producing regulatory T cells. Administration of DHEA in symptomatic mice induced regulatory CD4(+) T cells that were suppressive in an IL-10-dependent manner. Expression of the estrogen receptor ß by CD4(+) T cells was necessary for DHEA-mediated EAE amelioration, as well as for direct downregulation of Th17 responses. TGF-ß1 as well as aryl hydrocarbon receptor activation was necessary for the expansion of IL-10-producing T cells by DHEA. Thus, our studies demonstrate that compounds that inhibit pathogenic Th17 responses and expand functional regulatory cells could serve as therapeutic agents for autoimmune diseases, such as MS.

ERß in CD4+ T Cells Is Crucial for Ligand-Mediated Suppression of Central Nervous System Autoimmunity.

The development of therapies for multiple sclerosis targeting pathogenic T cell responses remains imperative. Previous studies have shown that estrogen receptor (ER) ß ligands could inhibit experimental autoimmune encephalomyelitis. However, the effects of ERß-specific ligands on human or murine pathogenic immune cells, such as Th17, were not investigated. In this article, we show that the synthetic ERß-specific ligand 4-(2-phenyl-5,7-bis[trifluoromethyl]pyrazolo[1,5-a]pyrimidin-3-yl)phenol (PHTPP) reversed established paralysis and CNS inflammation, characterized by a dramatic suppression of pathogenic Th responses as well as induction of IL-10-producing regulatory CD4(+) T cell subsets in vivo. Moreover, administration of PHTPP in symptomatic mice induced regulatory CD4(+) T cells that were suppressive in vivo. PHTPP-mediated experimental autoimmune encephalomyelitis amelioration was canceled in mice with ERß-deficient CD4(+) T cells only, indicating that expression of ERß by these cells is crucial for the observed therapeutic effect. Importantly, synthetic ERß-specific ligands acting directly on CD4(+) T cells suppressed human and mouse Th17 cells, downregulating Th17 cell signature gene expression and expanding IL-10-producing T cells among them. TGF-ß1 and aryl hydrocarbon receptor activation enhanced the ERß ligand-mediated expansion of IL-10-producing T cells among Th17 cells. In addition, these ERß-specific ligands promoted the induction and maintenance of Foxp3(+) T regulatory cells, as well as their in vitro suppressive function. Thus, ERß-specific ligands targeting pathogenic Th17 cells and inducing functional regulatory cells represent a promising subset of therapeutic agents for multiple sclerosis.

Effects of Prepubertal or Adult Site-Specific Knockdown of Estrogen Receptor ß in the Medial Preoptic Area and Medial Amygdala on Social Behaviors in Male Mice.

Testosterone, after being converted to estradiol in the brain, acts on estrogen receptors (ERα and ERß) and controls the expression of male-type social behavior. Previous studies in male mice have revealed that ERα expressed in the medial preoptic area (MPOA) and medial amygdala (MeA) are differently involved in the regulation of sexual and aggressive behaviors by testosterone action at the time of testing in adult and/or on brain masculinization process during pubertal period. However, a role played by ERß in these brain regions still remains unclear. Here we examined the effects of site-specific knockdown of ERß (ßERKD) in the MPOA and MeA on male social behaviors with the use of adeno-associated viral mediated RNA interference methods in ICR/Jcl mice. Prepubertal ßERKD in the MPOA revealed that continuous suppression of ERß gene expression throughout the pubertal period and adulthood decreased aggressive but not sexual behavior tested as adults. Because ßERKD in the MPOA only in adulthood did not affect either sexual or aggressive behaviors, it was concluded that pubertal ERß in the MPOA might have an essential role for the full expression of aggressive behavior in adulthood. On the other hand, although neither prepubertal nor adult ßERKD in the MeA had any effects on sexual and aggressive behavior, ßERKD in adulthood disrupted sexual preference of receptive females over nonreceptive females. Collectively, these results suggest that ERß in the MPOA and MeA are involved in the regulation of male sexual and aggressive behavior in a manner substantially different from that of ERα.

Estrogen receptor 2b deficiency impairs the antiviral response of zebrafish.

Although several studies have demonstrated the ability of some endocrine disruptive chemicals (EDCs) to alter the physiology of zebrafish, the immune-reproductive interaction has received little attention in this species. In this study, we used a homozygous line carrying an insertion of 8 amino acids in the ligand-binding domain of the estrogen receptor 2b gene (esr2b) to further understand the role of estrogen signaling on innate immunity. Adult mutant fish showed distorted sexual ratios related with alterations in testicular morphology and supraphysiological testosterone and 17ß-estradiol (E2) levels. Immunity-wise, although esr2b mutant fish showed unaltered antibacterial responses, they were unable to mount an effective antiviral response upon viral challenge. RT-qPCR analysis demonstrated that mutant fish were able to induce the genes encoding major antiviral molecules, including Ifnphi1, Ifnphi2, Infphi3, Mxb and Mxc, and the negative feedback regulator of cytokine signaling Socs1. Notably, although esr2b mutant larvae showed a similar resistance to SVCV infection to their wild type siblings, waterborne E2 increased their viral susceptibility. Similarly, the exposure of adult wild type zebrafish to E2 also resulted in increased susceptibility to SVCV infection. Finally, the administration of recombinant Ifnphi1 hardly reversed the higher viral susceptibility of esr2b mutant zebrafish, suggesting that elevated socs1 levels impair Ifn signaling. All together, these results uncover an important role for E2 and Esr signaling in the fine-tuning of sexual hormone balance and the antiviral response of vertebrates.

Neurite outgrowth promoting effect of 17-ß estradiol is mediated through estrogen receptor alpha in an olfactory epithelium culture.

Olfactory deficits are observed early in the course of chronic neurological disorders including Alzheimer's disease (AD). Estrogen treatment in post-menopausal women reduced the incidence of olfactory dysfunction, raising the possibility that estrogen treatment can cure olfactory deficits in preclinical stages of AD. In this study, we examined the estradiol׳s effects on neurite outgrowth in explant cultures of mouse olfactory epithelium (OE). We found that neurons in OE cultures treated with 100 pM 17-ß estradiol (estradiol) had significantly longer neurite outgrowth than cultures treated with ethanol alone (vehicle). The OE neurons expressed estrogen receptors alpha (ERα) and ER beta (ERß). Estrogen treatment upregulated both ERα and ERß expression in OE culture. Treatment of OE cultures with propyl pyrazole triol (PPT), a selective agonist for ERα increased neurite outgrowth to comparable extent as estradiol treatment. In contrast, 2,3-bis-4-hydroxyphenyl (DPN), a specific agonist for ERß, had no effect on neurite outgrowth. Furthermore, estradiol treatment increased neurite outgrowth in OE cultures derived from ERß-deficient/knockout mice and wild-type littermates, but not in ERα-deficient/knockout mice. These data suggest that ERα mediates the neurite outgrowth promoting effects of estradiol in OE cultures. We propose that olfactory dysfunction in chronic neurological disorders, where estrogen deficiency is a risk factor, is an indicator of compromised axonal regeneration of olfactory sensory neurons.

Estrogen receptor beta impacts hormone-induced alternative mRNA splicing in breast cancer cells.

BACKGROUND: Estrogens play an important role in breast cancer (BC) development and progression; when the two isoforms of the estrogen receptor (ERα and ERß) are co-expressed each of them mediate specific effects of these hormones in BC cells. ERß has been suggested to exert an antagonist role toward the oncogenic activities of ERα, and for this reason it is considered an oncosuppressor. As clinical evidence regarding a prognostic role for this receptor subtype in hormone-responsive BC is still limited and conflicting, more knowledge is required on the biological functions of ERß in cancer cells. We have previously described the ERß and ERα interactomes from BC cells, identifying specific and distinct patterns of protein interactions for the two receptors. In particular, we identified factors involved in mRNA splicing and maturation as important components of both ERα and ERß pathways. Guided by these findings, here we performed RNA sequencing to investigate in depth the differences in the early transcriptional events and RNA splicing patterns induced by estradiol in cells expressing ERα alone or ERα and ERß. RESULTS: Exon skipping was the most abundant splicing event in the post-transcriptional regulation by estradiol. We identified several splicing events induced by ERα alone and by ERα+ERß, demonstrating for the first time that ERß significantly affects estrogen-induced splicing in BC cells, as revealed by modification of a subset of ERα-dependent splicing by ERß, as well as by the presence of splicing isoforms only in ERß+cells. In particular, we observed that ERß+BC cell lines exhibited around 2-fold more splicing events than the ERß- cells. Interestingly, we identified putative direct targets of ERß-mediated alternative splicing by correlating the genomic locations of ERß and ERα binding sites with estradiol-induced differential splicing in the corresponding genes. CONCLUSIONS: Taken together, these results demonstrate that ERß significantly affects estrogen-induced early transcription and mRNA splicing in hormone-responsive BC cells, providing novel information on the biological role of ERß in these tumors.

Urethral dysfunction in female mice with estrogen receptor ß deficiency.

Estrogen has various regulatory functions in the growth, development, and differentiation of the female urogenital system. This study investigated the roles of ERß in stress urinary incontinence (SUI). Wild-type (ERß(+/+)) and knockout (ERß(-/-)) female mice were generated (aged 6-8 weeks, n = 6) and urethral function and protein expression were measured. Leak point pressures (LPP) and maximum urethral closure pressure (MUCP) were assessed in mice under urethane anesthesia. After the measurements, the urethras were removed for proteomic analysis using label-free quantitative proteomics by nano-liquid chromatography-mass spectrometry (LC-MS/MS) analysis. The interaction between these proteins was further analysed using MetaCore. Lastly, Western blot was used to confirm the candidate proteins. Compared with the ERß(+/+) group, the LPP and MUCP values of the ERß(-/-) group were significantly decreased. Additionally, we identified 85 differentially expressed proteins in the urethra of ERß(-/-) female mice; 57 proteins were up-regulated and 28 were down-regulated. The majority of the ERß knockout-modified proteins were involved in cell-matrix adhesion, metabolism, immune response, signal transduction, nuclear receptor translational regelation, and muscle contraction and development. Western blot confirmed the up-regulation of myosin and collagen in urethra. By contrast, elastin was down-regulated in the ERß(-/-) mice. This study is the first study to estimate protein expression changes in urethras from ERß(-/-) female mice. These changes could be related to the molecular mechanism of ERß in SUI.

The activation of G protein-coupled receptor 30 (GPR30) inhibits proliferation of estrogen receptor-negative breast cancer cells in vitro and in vivo.

There is an urgent clinical need for safe and effective treatment agents and therapy targets for estrogen receptor negative (ER-) breast cancer. G protein-coupled receptor 30 (GPR30), which mediates non-genomic signaling of estrogen to regulate cell growth, is highly expressed in ER--breast cancer cells. We here showed that activation of GPR30 by the receptor-specific agonist G-1 inhibited the growth of ER--breast cancer cells in vitro. Treatment of ER--breast cancer cells with G-1 resulted in G2/M-phase arrest, downregulation of G2-checkpoint regulator cyclin B, and induction of mitochondrial-related apoptosis. The G-1 treatment increased expression of p53 and its phosphorylation levels at Serine 15, promoted its nuclear translocation, and inhibited its ubiquitylation, which mediated the growth arrest effects on cell proliferation. Further, the G-1 induced sustained activation and nuclear translocation of ERK1/2, which was mediated by GPR30/epidermal growth factor receptor (EGFR) signals, also mediated its inhibition effects of G-1. With extensive use of siRNA-knockdown experiments and inhibitors, we found that upregulation of p21 by the cross-talk of GPR30/EGFR and p53 was also involved in G-1-induced cell growth arrest. In vivo experiments showed that G-1 treatment significantly suppressed the growth of SkBr3 xenograft tumors and increased the survival rate, associated with proliferation suppression and upregulation of p53, p21 while downregulation of cyclin B. The discovery of multiple signal pathways mediated the suppression effects of G-1 makes it a promising candidate drug and lays the foundation for future development of GPR30-based therapies for ER- breast cancer treatment.

Deficiency of ERß and prostate tumorigenesis in FGF8b transgenic mice.

Estrogens contribute to the development and growth of the prostate and are implicated in prostate tumorigenesis. In their target tissues, estrogens mediate their effects via estrogen receptor α (ERα (ESR1)) and ß (ERß (ESR2)). Hyperplasia and decreased differentiation of epithelial cells in the prostate have been reported in ERß knockout (BERKO) mice. Herein, we studied the effect of ERß deficiency on prostate tumorigenesis by crossing BERKOFVB mice with prostate-targeted human fibroblast growth factor 8b transgenic (FGF8b-Tg) mice. Consistent with results described in our previous report, the prostates of 1-year-old FGF8b-Tg mice displayed stromal aberrations, prostatic intraepithelial neoplasia (mPIN) lesions, inflammation, and occasionally cancer. The prostates of BERKOFVB mice exhibited mild epithelial hypercellularity and inflammation. The prostate phenotypes of FGF8b-Tg-BERKOFVB mice closely resembled those of FGF8b-Tg mice. However, mucinous metaplasia, indicated by Goblet-like cells in the epithelium, was significantly more frequent in the prostates of FGF8b-Tg-BERKOFVB mice when compared with FGF8b-Tg mice. Furthermore, compared with FGF8b-Tg mice, there was a tendency for increased frequency of inflammation but milder hyperplasias in the prostate stroma of FGF8b-Tg-BERKOFVB mice. The expression levels of mRNAs for FGF8b-regulated genes including osteopontin (Spp1), connective tissue growth factor (Ctgf), fibroblast growth factor receptors (Fgfrs), and steroid hormone receptors and cytokines were similar in the prostates of FGF8b-Tg and FGF8b-Tg-BERKOFVB mice. Our results indicate that ERß plays a role in the differentiation of the prostatic epithelium and, potentially, in the defensive mechanism required for protection against inflammation but do not support a direct tumor-suppressive function of ERß in the prostate of FGF8b-Tg mice.

The role of estrogen signaling in a mouse model of inflammatory bowel disease: a Helicobacter hepaticus model.

The pathogenesis of inflammatory bowel diseases (IBD), Crohn's disease and ulcerative colitis, is due in part to interactions between the immune system, genetics, the environment, and endogenous microbiota. Gonadal sex hormones (GSH), such as estrogen, are thought to be involved in the development of IBD as variations in disease severity occur during pregnancy, menopause, or oral contraceptives use. In certain strains of mice, infection with Helicobacter hepaticus triggers IBD-like mucosal inflammation that is more severe in female mice than in males, suggesting a role for GSH in this model. To determine the role of estrogen signaling in microbiota-induced intestinal inflammation, estrogen receptor (ER) α and ß knock-out (KO) mice, ER agonists, and adoptive transfers were utilized. We demonstrate that, when signaling is limited to ERß on a non-CD4+ cell subset, disease is less severe and this correlates with decreased expression of pro-inflammatory mediators.

Estrogen modulates cardiac growth through an estrogen receptor α-dependent mechanism in healthy ovariectomized mice.

The modulation of cardiac growth by estrogen in healthy mice is not completely understood. The aim was to investigate the effects of estrogen on cardiac growth in healthy mice lacking either estrogen receptor (ER) α or ß. Wild-type (WT), ERα knockout (ERKO) and ERß knockout (BERKO) 2-month-old mice were ovariectomized and randomly assigned to groups receiving an estradiol (E2)-containing or soy-free (control, CON) diet (n=5-7/group). After three months of E2 administration, WT and BERKO mice had significantly lower body weight, higher relative uterus and heart weight than CON mice, while there was no major E2 effect in ERKO mice. Furthermore, there was a higher concentration of E2-responsive genes Igf1 and Myocd in WT and BERKO but not in ERKO mice. Together, these findings indicate that the estrogenic regulation of cardiac growth in healthy mice is primarily mediated through ERα and not ERß.

The cross-talk between estrogen receptor and peroxisome proliferator-activated receptor gamma in thyroid cancer.

BACKGROUND: Estrogen receptor (ER) and peroxisome proliferator-activated receptor gamma (PPARγ) are associated with thyroid tumorigenesis and treatment. However, the interaction between them has not been studied. METHODS: The impact of ER over-expression or down-expression by DNA/small interfering RNA (siRNA) transfection, ERα agonists, and the ERß agonist diarylpropiolnitrile (DPN) on PPARγ expression/activity was examined in papillary thyroid carcinoma (PTC) and anaplastic thyroid carcinoma (ATC) cells. The effects of PPARγ modulation by rosiglitazone (RTZ), a PPARγ ligand, and of PPARγ siRNA on ER expression were determined. Cellular functions reflected by cell proliferation and migration were assayed. Apoptosis was analyzed by terminal deoxynucleotidyl transferase dUTP nick-end labeling, and apoptotic-related proteins were evaluated by Western blot analysis. RESULTS: PPARγ protein and activity were reduced by the over-expression of either ERα or ERß, whereas repression of ERα or ERß increased PPARγ expression. The administration of RTZ counteracted the effects of ER and also reduced their expression, particularly in PTC cells. Moreover, knockdown of PPARγ increased ER expression and activity. Functionally, ERα activation offset the inhibitory effect of PPARγ on cellular functions, but ERß activation aggregated it and induced apoptosis, particularly in PTC cells. Finally, the interaction between ERß and PPARγ enhanced the expression of proapoptotic molecules, such as caspase-3 and apoptosis-inducing factor. CONCLUSIONS: This study provides evidence supporting a cross-talk between ER and PPARγ. The reciprocal interaction between PPARγ and ERß significantly inhibits the proliferation and migration of thyroid cancer cells, providing a new therapeutic strategy against thyroid cancer.

Sex- and estrogen-dependent regulation of a miRNA network in the healthy and hypertrophied heart.

BACKGROUND: In pressure overload, profibrotic gene expression and cardiac fibrosis are more pronounced in males than in females. Sex-specific and estrogen-dependent regulation of microRNAs (miRNAs), such as miR-21, may be a potential mechanism leading to sex differences in fibrosis. OBJECTIVES: To analyze the influence of sex, estrogen, and estrogen receptor beta (ERß) on the expression of miR-21 and to identify additional miRNAs potentially involved in sex-specific pressure overload-induced cardiac remodeling. METHODS: The sex-specific regulation of fibrosis-related miRNAs was analyzed in male and female wild type and ERß-deficient mice after transverse aortic constriction (TAC), in rat fibroblasts, and in a cardiomyocyte-like cell line. RESULTS: We report the sex-specific expression of functionally-related miR-21, -24, -27a, -27b, 106a, -106b and the regulation of their expression by estrogen in a sex-specific manner. These effects were abolished in ERß-deficient mice. We demonstrate the presence of common functional target sites for these miRNAs on three repressors of the mitogen-activated protein kinase signaling pathway, i.e. Rasa1, Rasa2 and Spry1, which may all lead to cardiac fibrosis. As expected, transfection with miRNA mimics targeting these repressors induced ERK1/2 phosphorylation. CONCLUSIONS: Estrogen regulates a network of miRNAs in a sex-specific manner via ERß. Our data suggest that the sex-specific expression of these miRNAs may be related to sex differences in fibrosis after pressure overload.