OBJECTIVEAIM To establish a drug screening model based on transcriptional regulation of estrogen responsive element (ERE) and use it to screen compounds for discovering new ligands of estrogen receptor (ER) subtypes.

METHODA recombinant reporter vector pERE-TAL-SEAP was constructed by inserting a synthetic sequence composed of five tandem copies of EREs upstream of promoter of the reporter vector pTAL-SEAP. The pERE-TAL-SEAP and the internal control plasmid pCMV were transiently co-transfected into Hela cells expressing ER subtype or ER subtype, and the effects of pure ER agonists 17estradiol, phytoestrogen genistein and pure ER antagonist ICI182, 780 on reporter gene SEAP expression were observed.

RESULTIn the Hela cells expressing ER alpha or ER beta subtype, the expression of SEAP gene were induced in a dose dependent manner by 17-estrodiol with a maximal effect at approximately 10 nmol.L-1 and with EC50 of (80.58 +/- 8.51) pmol.L-1 and (103.90 +/- 5.29) pmol.L-1, respectively, so done by phytoestrogen genistein with a maximal effect at 1 mumol.L-1 and with EC50 of (10.86 +/- 0.75) nmol.L-1 and (39.38 +/- 2.26) nmol.L-1, respectively. The maximal level induced by estrodiol and genistein were about 7-14 fold higher than that of vehicle. The pure antiestrogen ICI182, 780 at concentration of 1 mumol.L-1 completely blocked the inductions of 17-estrodiol and genistein.

CONCLUSIONThe cellular drug screening model can be established by transfecting reporter vector pERE-TAL-SEAP in Hela cell lines expressing ER alpha or ER beta. The cell lines can be used to screen compounds with estrogenicity by testing SEAP activity in the culture media of cells growing in microtitier wells. The system should provide an efficient model for screening and analyzing the activity of large numbers of ligands of ER.

Drug Evaluation, Preclinical ; methods ; Estradiol ; pharmacology ; Estrogen Receptor alpha ; Estrogen Receptor beta ; Gene Expression Regulation ; drug effects ; Genes, Reporter ; Genistein ; pharmacology ; HeLa Cells ; Humans ; Ligands ; Promoter Regions, Genetic ; Receptors, Estrogen ; genetics ; Transfection

Estrogen plays an important role in the regulation of male reproduction. Through binding with the estrogen receptor (ER), estrogen produces genomic and non-genomic effects. Estrogen receptors include ERalpha and ERbeta which distribute in the male reproductive system including the testis, epididymis, prostate and penis. The spermatogenesis is impaired in mice with ERalpha gene knockout; however, it remains normal in mice with ERbeta gene knockout. This phenomenon suggests that the two subtypes of ER play different roles in spermatogenesis. Moreover, ERalpha or ERbeta may also act as a substitute of another.
Animals ; Estrogen Receptor alpha ; metabolism ; Estrogen Receptor beta ; metabolism ; Genitalia, Male ; metabolism ; Male ; Mice ; Receptors, Estrogen ; metabolism

One third of estrogens in the male are from the testis and the others from outside the testis. Aromatase P450 (CYP19) is an enzyme responsible for the conversion of androgens to estrogens. Estrogens regulate cell function via specific receptors--estrogen receptors (ER) which include ER alpha and ER beta. It has been found that the role of estrogens in male reproduction is complex and important, particularly during the neonatal life. Males lacking ER alpha are completely infertile because ER alpha-induced estrogens regulate the reabsorption of luminal fluid in the head of the epididymis and disruption of this essential function causes sperm to enter the epididymis diluted, rather than concentrated, resulting in infertility. Whereas males lacking aromatase or ER beta are fully fertile. Therefore, it is concluded that ER alpha, but not aromatase or ER beta, is essenitial for normal male fertility.
Animals ; Aromatase ; physiology ; Estrogen Receptor alpha ; Estrogen Receptor beta ; Estrogens ; physiology ; Humans ; Male ; Receptors, Estrogen ; physiology ; Reproduction ; physiology

OBJECTIVESTo study extracellular signal-regulated kinase (ERK) activation in the endometrial carcinoma cell line Ishikawa with stimulation by 17-beta-estradiol, and to elucidate the role of phosphatase and tensin homologue (PTEN) and estrogen receptor (ER) subtype on the activation of ERKs.

METHODSWestern blot was used to examine the expression of PTEN and PTEN (G129E) in Ishikawa cells after stable transfection as well as ERK activation in Ishikawa-EGFP, Ishikawa- PTEN and Ishikawa- PTEN (G129E) stimulated with various doses of 17-beta-estradiol for different lengths of time. Western blot was also used for examining the expression of ERalpha and ERbeta in NIH3T3 fibroblasts after transient transfection of pCXN2hERalpha and pCXN2hERbeta. Then, ERK activation was examined after stimulation with 17-beta-estradiol.

RESULTS17-beta-estradiol activated ERK cascades (mainly ERK2) in Ishikawa cells. The activation of ERK increased gradually as concentration of 17-beta-estradiol also increased. The maximal activation of ERK2 took place 5 min after stimulation with 17-beta-estradiol. The activation of ERK2 was inhibited markedly by PTEN, but not by PTEN (G129E). 17-beta-estradiol activated ERK cascades in NIH3T3 fibroblasts after transient transfection of pCXN2hERalpha.

CONCLUSIONS17-beta-estradiol activate ERK cascades in Ishikawa cells by integrating with ERalpha. Lipid phosphatase PTEN has an inhibitory role on the activation of ERK stimulated by 17-beta-estradiol in Ishikawa cells.

3T3 Cells ; Animals ; Endometrial Neoplasms ; enzymology ; Enzyme Activation ; Estradiol ; pharmacology ; Estrogen Receptor alpha ; Estrogen Receptor beta ; Female ; Mice ; Mitogen-Activated Protein Kinases ; metabolism ; PTEN Phosphohydrolase ; Phosphoric Monoester Hydrolases ; genetics ; physiology ; Receptors, Estrogen ; physiology ; Tumor Cells, Cultured ; Tumor Suppressor Proteins ; genetics ; physiology

PURPOSE: In our previous studies we showed that upregulating claudin-6 (CLDN6) expression may contribute to preventing breast cancer, and that 17beta-estradiol induces a concentration- and time-related effect on CLDN6 mRNA and protein expression in MCF-7 cells. However, the mechanisms of 17beta-estradiol regulation of CLDN6 are still unclear. We determined the role of estrogen receptors in the regulation of CLDN6 expression in human breast cancer tissues and a cell line. METHODS: CLDN6, estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta) expression in breast cancer tissues were examined using immunohistochemistry. The human breast cancer cell line, MCF-7, which expresses ERalpha but not ERbeta was used. CLDN6 and ERalpha expression were measured by reverse transcriptase-PCR, Western blotting and immunofluorescent staining. Treatments with propyl pyrazole triol (PPT) and ICI 182, 780 (ICI) were performed. RESULTS: The results revealed that CLDN6 expression was related to ERalpha in breast cancer tissues (p=0.033). PPT, an ERalpha-selective ligand, upregulated CLDN6 expression at 10-5 mol/L after 24 hours. The effect of PPT on regulating CLDN6 expression in MCF-7 cells was blocked by ICI. CONCLUSION: These findings suggest that Eralpha reulates CLDN6 expression in breast cancer tissues and that 17beta-estradiol induces CLDN6 expression through an ERalpha pathway in MCF-7 cells.
Blotting, Western ; Breast ; Breast Neoplasms ; Cell Line ; Claudins ; Estrogen Receptor alpha ; Estrogen Receptor beta ; Estrogens ; Humans ; Immunohistochemistry ; MCF-7 Cells ; Pyrazoles ; Receptors, Estrogen ; RNA, Messenger ; Tight Junctions

Estrogens, a class of steroid hormones, regulate the growth, development, and physiology of the human reproductive system. Estrogens also involve in the neuroendocrine, skeletal, adipogenesis, and cardiovascular systems. Estrogen signaling pathways are selectively stimulated or inhibited depending on a balance between the activities of estrogen receptor (ER) alpha or ERbeta in target organs. ERs belong to the steroid hormone superfamily of nuclear receptors, which act as transcription factors after binding to estrogen. The gene expression regulation by ERs is to modulate biological activities, such as reproductive organ development, bone modeling, cardiovascular system functioning, metabolism, and behavior in both females and males. Understanding of the general physiological roles of ERs has been gained when estrogen levels were ablated by ovariectomy and then replenished by treatment with exogenous estrogen. This technique is not sufficient to fully determine the exact function of estrogen signaling in general processes in living tissues. However, a transgenic mouse model has been useful to study gene-specific functions. ERalpha and ERbeta have different biological functions, and knockout and transgenic animal models have distinct phenotypes. Analysis of ERalpha and ERbeta function using knockout mouse models has identified the roles of estrogen signaling in general physiologic processes. Although transgenic mouse models do not always produce consistent results, they are the useful for studying the functions of these genes under specific pathological conditions.
Adipogenesis ; Animals ; Animals, Genetically Modified ; Cardiovascular System ; Estrogen Receptor alpha ; Estrogen Receptor beta ; Estrogens ; Female ; Gene Expression Regulation ; Humans ; Male ; Mice ; Mice, Knockout ; Mice, Transgenic ; Ovariectomy ; Phenotype ; Receptors, Cytoplasmic and Nuclear ; Receptors, Estrogen ; Transcription Factors

OBJECTIVETo observe the effects of low-dose exogenous estrogen nonylphenol (NP) on the proliferation of human prostate cancer cell lines DU-145 and the expression of the membrane estrogen receptor GPR30 in the DU-145 cells.

METHODSWe exposed DU-145 cells to different concentrations of NP for 24 hours, followed by measurement of the half maximal inhibitory concentration (IC50) of the cells by cell proliferation assay and determination of the concentration of exposure to low-dose NP. We also observed the expressions of 3 estrogen receptors (ER), including ER-alpha, ER-beta and membrane estrogen receptor GPR30, in the DU-145 cells exposed to low-dose NP by RT-PCR.

RESULTSCell proliferation assay showed that within a certain range of doses, NP inhibited the proliferation of the DU-145 cells with an IC50 of 46 micromol/L, a much lower dose of NP than IC50, 0.01, 0.1.1 micromol/l NP, that can promote the proliferation of DU-145 cells. The results of RT-PCR indicated that the expressions of the three ERs in the DU-145 cells were similar to those in prostate epithelial cells, and that low-dose NP promoted the expression of GPR30.

CONCLUSIONMembrane estrogen receptor GPR30 may play a role in low-dose NP promoting the proliferation of DU-145 cells.

Cell Line, Tumor ; Cell Proliferation ; drug effects ; physiology ; Estrogen Receptor alpha ; metabolism ; Estrogen Receptor beta ; metabolism ; Estrogens ; Humans ; Male ; Phenols ; administration & dosage ; pharmacology ; Prostatic Neoplasms ; metabolism ; pathology ; Receptors, Estrogen ; metabolism ; Receptors, G-Protein-Coupled ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction

PURPOSE: Estrogen receptor (ER) is the key therapeutic target in breast cancer. ERbeta has recently been identified to be distinct from ERalpha. In contrast to ERalpha, the functions of ERbeta in breast cancer are still unclear. We sought to determine whether the expression of ERbeta can be used as a predictive marker for endocrine therapy for patients with ERalpha-negative breast cancer. METHODS: Formalin-fixed, paraffin-embedded tumor specimens from 52 patients with ER-/PR+ invasive breast cancer were immunostained for their ERbeta expression. These patients were treated with adjuvant tamoxifen. The results were correlated with various clinicopathological variables and the follow-up data. The expressions of p53 and HER-2/neu were also analyzed and correlated with the ERbeta status. RESULTS: An ERbeta expression was observed in 53.8% (28/52) of the breast cancer samples. There was no correlation between the ERbeta expression and the other clinicopathologic factors (age, tumor size, histologic type, nodal status, histological grade, stage, therapeutic modality, progesterone receptor (PR) expression, p53 expression and HER-2/neu expression). Recurrence was present in 7.7% (2/26) of the patients whose tumors had an ERbeta expression, as compared to the presence of recurrence in 36.4% (8/22) of the patients whose tumors had no ERbeta expression (p<0.05). The patients with ERbeta negative-tumors revealed lower disease free survival rate than those with ERbeta positive-tumors (p<0.05). Of the 52 patients, 10 (19.2%) were p53 positive, and 11 (21.2%) were HER-2/neu positive. No significant correlations were observed between ERbeta and p53 or HER-2/neu. CONCLUSION: These results suggest that ERbeta might be a predictive marker of a response to endocrine therapy in patients with ER-/PR+ invasive breast cancer, although this needs to be confirmed by additional studies.
Breast ; Breast Neoplasms ; Disease-Free Survival ; Estrogen Receptor alpha ; Estrogen Receptor beta ; Estrogens ; Follow-Up Studies ; Humans ; Progesterone ; Receptors, Progesterone ; Recurrence ; Tamoxifen

Asian Continental Ancestry Group ; genetics ; Bone Density ; Estrogen Receptor alpha ; genetics ; Estrogen Receptor beta ; genetics ; Humans ; Osteoporosis ; genetics ; Receptors, Calcitriol ; genetics

OBJECTIVETo investigate the distribution of ER isoforms in endometriosis and eutopic endometrium.

METHODSTissue samples of patients with ovarian endometriosis, treated in People's Liberation Army General Hospital from January 2004 to December 2006, were retrieved. A total of 60 cases of ovarian endometriotic cysts with their corresponding eutopic endometrium (30 cases of proliferation phase and 30 of secretary phase eutopic endometrium) and 30 cases of normal endometrium (15 proliferative and 15 secretary phase endometrial samples respectively) were included. Expressions of ERalpha and ERbeta were analyzed using immunohistochemistry and the expression ratio was statistically analyzed by using SPSS 12.0 software.

RESULTSExpressions of both ERalpha and ERbeta in epithelial cells were positively correlated with that of the stromal cells. The expression of ERalpha in eutopic endometrium (73.3% in epithelium and 76.7% in stroma) was significantly higher than that in ovarian endometriotic cysts (43.3% in epithelium and 46.7% in stroma), or normal control (56.7% in epithelium and 50.0% in stroma, respectively, each P < 0.05. However, the expression of ERbeta (90.0% in epithelium and 76.7% in stroma) was higher in ovarian endometriotic cysts than that in the eutopic endometrium (68.0% in epithelium and 63.3% in stroma respectively, P < 0.05), and ERbeta expression in eutopic endometrium was higher than that in the normal control endometrium (36.7% in epithelium and 26.7% in stroma, respectively, P < 0.05). The expressions of both ERalpha and ERbeta changed periodically in eutopic and normal endometrium, whereas ERalpha and ERbeta level were less variable in the ectopic endometrium. The expression of ERbeta was statistically higher than that of ERalpha (P < 0.05) in ectopic endometrium, whereas no significant difference was seen between the two isoforms in the eutopic or normal endometrium.

CONCLUSIONSBoth ERalpha and ERbeta have higher expression levels in eutopic endometrium of patients with ovarian endometriotic cysts. ERbeta is predominantly expressed in endometriotic cysts, where the expression of ERalpha is limited. The different distribution of ERalpha and ERbeta may play an important role in the development of ovarian endometriosis.

Adult ; Choristoma ; pathology ; Endometriosis ; metabolism ; pathology ; Endometrium ; metabolism ; pathology ; Epithelium ; Estrogen Receptor alpha ; analysis ; Estrogen Receptor beta ; analysis ; Female ; Humans ; Immunohistochemistry ; Middle Aged ; Protein Isoforms ; analysis ; Receptors, Estrogen ; analysis ; Stromal Cells ; metabolism