BackgroundEndometrial cancer is the most common gynaecological malignancy; risk factors include exposure to oestrogens and high body mass index. Expression of enzymes involved in biosynthesis of oestrogens and prostaglandins (PG) is often higher in endometrial cancers when compared with levels detected in normal endometrium. Oestrogens bind one of two receptors (ERα and ERβ) encoded by separate genes. The full-length receptors function as ligand-activated transcription factors; splice variant isoforms of ERβ lacking a ligand-binding domain have also been described. PGs act in an autocrine or paracrine manner by binding to specific G-protein coupled receptors.MethodsWe compared expression of ERs, progesterone receptor (PR) and cyclooxygenase-2 (COX-2) in stage 1 endometrial adenocarcinomas graded as well (G1), moderately (G2) or poorly (G3) differentiated (n ≥ 10 each group) using qRTPCR, single and double immunohistochemistry. We used endometrial adenocarcinoma cell lines to investigate the impact of PGF2α on expression of ERs and PR.ResultsFull length ERβ (ERβ1) and two ERβ variants (ERβ2, ERβ5) were expressed in endometrial cancers regardless of grade and the proteins were immunolocalised to the nuclei of cells in both epithelial and stromal compartments. Immunoexpression of COX-2 was most intense in cells that were ERαneg/low. Expression of PR in endometrial adenocarcinoma (Ishikawa) cell lines and tissues broadly paralleled that of ERα. Treatment of adenocarcinoma cells with PGF2α reduced expression of ERα but had no impact on ERβ1. Cells incubated with PGF2α were unable to increase expression of PR mRNA when they were incubated with E2.ConclusionWe have demonstrated that ERβ5 protein is expressed in stage 1 endometrial adenocarcinomas. Expression of three ERβ variants, including the full-length protein is not grade-dependent and most cells in poorly differentiated cancers are ERβpos/ERαneg. We found evidence of a link between COX-2, its product PGF2α, and expression of ERα and PR that sheds new light on the cross talk between steroid and PG signalling pathways in this disease.
Context:The endometrium is a multicellular, steroid-responsive tissue that undergoes dynamic remodeling every menstrual cycle in preparation for implantation and, in absence of pregnancy, menstruation. Androgen receptors are present in the endometrium.Objective:The objective of the study was to investigate the impact of androgens on human endometrial stromal cells (hESC).Design:Bioinformatics was used to identify an androgen-regulated gene set and processes associated with their function. Regulation of target genes and impact of androgens on cell function were validated using primary hESC.Setting:The study was conducted at the University Research Institute.Patients:Endometrium was collected from women with regular menses; tissues were used for recovery of cells, total mRNA, or protein and for immunohistochemistry.Results:A new endometrial androgen target gene set (n = 15) was identified. Bioinformatics revealed 12 of these genes interacted in one pathway and identified an association with control of cell survival. Dynamic androgen-dependent changes in expression of the gene set were detected in hESC with nine significantly down-regulated at 2 and/or 8 h. Treatment of hESC with dihydrotestosterone reduced staurosporine-induced apoptosis and cell migration/proliferation.Conclusions:Rigorous in silico analysis resulted in identification of a group of androgen-regulated genes expressed in human endometrium. Pathway analysis and functional assays suggest androgen-dependent changes in gene expression may have a significant impact on stromal cell proliferation, migration, and survival. These data provide the platform for further studies on the role of circulatory or local androgens in the regulation of endometrial function and identify androgens as candidates in the pathogenesis of common endometrial disorders including polycystic ovarian syndrome, cancer, and endometriosis.
Endometriosis is an estrogen-dependent neurovascular disorder characterized by growth of endometrial tissue (lesions) outside the uterine cavity. Patients suffer chronic pelvic pain, and it has been proposed that co-recruitment of nerves/blood vessels (neuroangiogenesis) into the lesions is fundamental to the development of painful symptoms. We hypothesized that estrogen-dependent regulation of axonal guidance molecules of the SLIT/ROBO (Roundabout) family could play a role in neuroangiogenesis occurring in endometriosis lesions found on the peritoneal wall. In tissue samples from human patients and a mouse model of endometriosis, concentrations of mRNA encoded by SLIT3 were significantly higher in lesions than normal peritoneum. Estrogen regulation of SLIT3 was investigated using 17β-estradiol and selective agonists for each subtype of estrogen receptor (ER) (ERα agonist, 4,4',4″-(4-propyl-(1H)-pyrazole-1,3,5-tryl) trisphenol; ERβ agonist, 2,3-bis(4-hydroxy-phenyl)-propionitrile [DPN]). In mice, DPN (EC50 0.85) increased Slit3 mRNA concentrations compared with hormone-depleted and 17β-estradiol-treated (EC50 0.1) animals and decreased the density of nerves but not vessels in endometriosis lesions. SLIT3 mRNA concentrations were increased in DPN-treated human endometrial endothelial cells and in 4,4',4″-(4-propyl-(1H)-pyrazole-1,3,5-tryl) trisphenol-treated (EC50 200) rat dorsal root ganglia neurons. Functional assays (neurite outgrowth, network formation) revealed that SLIT3 promotes angiogenesis but decreases neurogenesis. In conclusion, these data suggest that estrogen-dependent expression of SLIT3 may play a key role in regulating nerve-vessel interactions within the complex microenvironment of endometriosis lesions.
Endometrial cancer (EC) and ovarian cancer are common gynaecological malignancies. The impact of androgen action in these cancers is poorly understood; however, there is emerging evidence to suggest that targeting androgen signalling may be of therapeutic benefit. Epidemiological evidence suggests that there is an increased risk of EC associated with exposure to elevated levels of androgens, and genetic variants in genes related to both androgen biosynthesis and action are associated with an increased risk of both EC and ovarian cancer. Androgen receptors (ARs) may be a potential therapeutic target in EC due to reported anti-proliferative activities of androgens. By contrast, androgens may promote growth of some ovarian cancers and anti-androgen therapy has been proposed. Introduction of new therapies targeting ARs expressed in EC or ovarian cancer will require a much greater understanding of the impacts of cell context-specific AR-dependent signalling and how ARs can crosstalk with other steroid receptors during progression of disease. This review considers the evidence that androgens may be important in the aetiology of EC and ovarian cancer with discussion of evidence for androgen action in normal and malignant endometrial and ovarian tissue.
STUDY QUESTIONWhat are the in vitro effects of estrogen receptor β (ERβ) activation on the function of endothelial cells (ECs) from different vascular beds: human endometrial ECs (HEECs; endometrium), uterine myometrial microvascular ECs (UtMVECs; myometrium) and human umbilical vein ECs (HUVECs)?SUMMARY ANSWERStudies conducted in vitro demonstrate that the ERβ agonist 2,3-bis(4-hydroxy-phenyl)-propionitrile (DPN) has EC type-specific effects on patterns of gene expression and network formation. Identification of a key role for the transcription factor Sp1 in ERβ-dependent signaling in uterine ECs offers new insights into cell-specific molecular mechanisms of estrogen action in the human uterus.WHAT IS KNOWN ALREADYEstrogens, acting via ERs (ERα and ERβ), have important, body-wide impacts on the vasculature. The human uterus is an estrogen target organ, the endometrial lining of which exhibits physiological, cyclical angiogenesis. In fixed tissue sections, human endometrial ECs are immunopositive for ERβ.STUDY DESIGN, SIZE, DURATIONCells were treated with a vehicle control or the ERβ agonist, DPN, for 2 h or 24 h (n = 5) followed by gene expression analysis. Functional assays were analyzed after a 16 h incubation with ligand (n = 5).PARTICIPANT/MATERIALS, SETTING, METHODSAnalysis of DPN-treated ECs using Taqman gene array cards focused on genes involved in angiogenesis and inflammation identified cell type-specific ERβ-dependent changes in gene expression, with validation using qPCR and immunohistochemistry. Molecular mechanisms involved in ERβ signaling were investigated using bioinformatics, reporter assays, immunoprecipitation, siRNA and a specific inhibitor blocking Sp1-binding sites. The endometrium and myometrium from women with regular menses were used to validate the protein expression of candidate genes.MAIN RESULTS AND THE ROLE OF CHANCEHEECs and UtMVECs were ERβ+/ERα−. Treatment of ECs with DPN had opposite effects on network formation: a decrease in network formation in HEECs (P ≤ 0.001) but an increase in UtMVECs (P ≤ 0.05). Genomic analysis identified opposite changes in ERβ target gene expression with only three common transcripts (HEY1, ICAM1, CASP1) in all three ECs; a unique profile was observed for each. An important role for Sp1 was identified, consistent with the regulation of ERβ target genes via association with the transcription factor (‘tethered’ mechanism).LIMITATIONS, REASONS FOR CAUTIONThe study was mainly carried out in vitro using ECs of which one type was immortalized. Although the analysis of the protein expression of candidate genes was carried out using intact tissue samples from patients, investigations into in vivo angiogenesis were not carried out.WIDER IMPLICATIONS OF THE FINDINGSThese results have implications for our understanding of the mechanisms responsible for ERβ-dependent changes in EC gene expression in hormone-dependent disorders.STUDY FUNDING/COMPETEING INTEREST(S)The study was funded by a Medical Research Council Programme Grant. E.G. is the recipient of an MRC Ca...
BACKGROUNDHuman embryo implantation is regulated by estradiol (E2), progesterone and locally produced mediators including interleukin-1β (IL-1β). Interactions between the estrogen receptor (ER) and NF kappa B (NFκB) signalling pathways have been reported in other systems but have not been detailed in human endometrium.METHODS AND RESULTSReal-time PCR showed that mRNA for the p65 and p105 NFκB subunits is maximally expressed in endometrium from the putative implantation window. Both subunits are localized in the endometrial epithelium throughout the menstrual cycle. Reporter assays for estrogen response element (ERE) activity were used to examine functional interactions between ER and NFκB in telomerase immortalized endometrial epithelial cells (TERT-EEC). E2 and IL-1β treatment of TERT-EECs enhances ERE activity by a NFκB and ER dependent mechanism; this effect could be mediated by ERα or ERβ. E2 and IL-1β also positively interact to increase endogenous gene expression of prostaglandin E synthase and c-myc. This is a gene-dependent action as there is no additive effect on cyclin D1 or progesterone receptor expression.CONCLUSIONIn summary, we have established that NFκB signalling proteins are expressed in normal endometrium and report that IL-1β can enhance the actions of E2 in a cell line derived from healthy endometrium. This mechanism may allow IL-1β, possibly from the developing embryo, to modulate the function of the endometrial epithelium to promote successful implantation, for example by regulating prostaglandin production. Aberrations in the interaction between the ER and NFκB signalling pathways may have a negative impact on implantation contributing to pathologies such as early pregnancy loss and infertility.
Levels of estrogen within the male reproductive tract are higher than in the general circulation and the aromatase enzyme is expressed in the adult testis. Estrogens such as estradiol (E2) modify cell function by binding to high-affinity estrogen receptors (ER). Two subtypes (ERalpha and ERbeta) have been identified. Studies in animals have shown that over- or underexposure to estrogens can have an impact on testis function. For example, mice with targeted disruption of the aromatase cyp19 gene become infertile because round spermatids fail to differentiate normally. In rodents, ERalpha is expressed in Leydig cells; ERalpha mRNA and protein are not detectable in testes from humans or primates. High levels of expression of ERalpha occur in the efferent ductules in rodents, primates, and the human. ERbeta protein has been immunolocalized to all somatic cells and to some germ cells in these same species. Messenger RNAs for splice variant isoforms of human ERbeta are expressed in human testes. Homologues of the ERbeta2 variant have been cloned from primates; this isoform does not exist in rodents and does not bind E2. Full-length ERbeta protein (ERbeta1) and ERbeta2 have differential patterns of expression in human testes. In conclusion, although estrogens are synthesized in the testis and it has been suggested that E2 may function as a germ cell survival factor, the mechanisms by which estrogens influence male fertility remain uncertain and rodents may be poor models in which to examine this.
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