ERalpha mRNA Research Articles

Role of the cross-talk between estrogen receptors and peroxisome proliferator-activated receptor gamma in daidzein's prevention and treatment of osteoporosis in ovariectomized rats

To investigate the role of the cross-talk between estrogen receptors and peroxisome proliferator-activated receptor gamma in daidzein's prevention and treatment of osteoporosis in the ovariectomized rats. Sixty four-month-old Sprague-Dawley (SD) female rats were divided into six groups. Four weeks after the establishment of osteoporosis model, the ovariectomized rats were administered different drugs by gavage respectively for three months. After sacrificing, the expressions of mRNA of ERalpha, ERbeta and PPARgamma in L(6) and right femur were measured by realtime-RT-PCR, while proteins were detected by immunohistochemistry. The expressions of ERalpha mRNA and protein were significantly lower than that of ERbeta in three dosing groups. Excepting that of rat femur of rats in H-Dai group, the expressions of ERbeta mRNA of lumbar spine and femur in M-Dai group (0.0177 +/- 0.0010, 0.0245 +/- 0.0013) and L-Dai groups (0.0276 +/- 0.0027, 0.0278 +/- 0.0044) and lumbar spine in H-Dai group (0.0163 +/- 0.0037) were significantly higher than that in OVX group (0.0016 +/- 0.0005, 0.0041 +/- 0.0014). There was no significant difference with E(2) group. At the same time, the expression of ERbeta protein showed into the similar trend as that of mRNA. And there was an rising expressions of ERbeta while daidzein dose decreased. On the contrary, the expressions of PPARgamma mRNA and protein decreased. There is an inverse cross-talk relationship between ERbeta and PPARgamma, it may play a role in daidzein's prevention and treatment of osteoporosis in ovariectomized rats.

Gli1 promotes cell survival and is predictive of a poor outcome in ERα-negative breast cancer

Gli1 is a transcription factor and oncogene with documented roles in the progression of several cancer types, including cancers of the skin and pancreas. The contribution of Gli1 to the progression of breast cancer is less established. In order to investigate the functional impact of Gli1 in breast cancer, expression of Gli1 and its contribution to cell growth was assessed in breast cancer cell lines. These in vitro results were compared to expression of Gli1, determined by immunohistochemistry, in 171 breast cancers. In these cancers, the association of Gli1 with expression of estrogen receptor alpha (ERalpha) and progesterone receptor (PR), ErbB2, p53, the rate of proliferation, and clinicopathologic parameters and outcome was assessed. Expression of Gli1 and ERalpha mRNA was strongly correlated in ERalpha-positive cell lines (r = 0.999). Treatment with estrogen increased expression of Gli1 in 2 of 3 ERalpha-positive cell lines; this increase was prevented by treatment with the ERalpha-specific antagonist MPP. Silencing of Gli1 by shRNA markedly reduced the survival of two ERalpha-negative cell lines, but caused only a modest reduction in ERalpha-positive cell lines. In breast cancer tissues, cancers with nuclear localization of Gli1 had a higher ERalpha (P=0.027) and lower p53 expression (P=0.017) than those without nuclear localization of Gli1. However, nuclear localization of Gli1 was predictive of a poorer cancer-specific survival in ERalpha-negative, including triple negative, cancers (P = 0.005), but not ERalpha-positive cancers. In conclusion, we demonstrate a positive association between expression of Gli1 and ERalpha; however, our data indicate a greater functional effect of Gli1 in ERalpha-negative cancers.

Effects of 17β-trenbolone in male eelpout Zoarces viviparus exposed to ethinylestradiol

To evaluate the interaction between 17beta-trenbolone (TB) and 17alpha-ethinylestradiol (EE2), male eelpout, Zoarces viviparus, was exposed for 21 days (April to May 2008) to 5 ng l(-1) EE2 and 5 or 20 ng l(-1) TB, separately or in combination in a flow-through SW system. The effects on hepatosomatic (HSI) and gonadosomatic index (GSI), plasma vitellogenin (Vtg) concentration, gonadal histology, hepatic and testicular Vtg mRNA and estrogen receptor (ERalpha) mRNA expression were investigated. No effects on HSI were observed. A significant decrease was observed in the GSI of all males exposed to EE2 (<0.7%) when compared to controls (1.4%). Histological alterations and immature stages were observed in the testis of all exposed males; however, males exposed to EE2 were the most affected. Increased tubule number and proportionally decreased tubule diameter were observed in the testis of all EE2 groups. No effects in Vtg mRNA expression were observed in the testis; however, a significant decrease in testis ERalpha mRNA was observed in males exposed to 20 ng l(-1) TB. The groups exposed to EE2 showed a significant increase in plasma Vtg (>300-fold), hepatic Vtg mRNA (>450-fold), and ERalpha mRNA (>100-fold) when compared to controls. This study shows that lower concentrations of 17beta-trenbolone are unable to counteract the EE2 estrogenic effects when the exposure is simultaneous.

Estrogen receptor alpha inhibits the estrogen-mediated suppression of HIV transcription in astrocytes: implications for estrogen neuroprotection in HIV dementia.

Many human immunodeficiency virus (HIV) proteins including Tat are produced by HIV-infected astrocytes and secreted into the brain resulting in extensive neuronal damage that contributes to the pathogenesis of HIV dementia. The neuroprotective hormone 17beta-estradiol (E2) is known to negatively regulate the HIV transcriptional promoter in human fetal astrocytes (SVGA cell line) in a Tat-dependent manner. In the present study we extended our investigation in HIV-infected SVGA cells and found a reduction in HIV p24 levels following E2 treatment in comparison to control. Although many E2-mediated events occur through estrogen receptor alpha (ERalpha), we found low levels of ERalpha mRNA and failed to detect ERalpha protein in SVGA cells. Paradoxically, when ERalpha was overexpressed the E2-mediated decrease in Tat transactivation of the promotor was prevented. To determine whether ERalpha expression is altered in the human brain following HIV infection, postmortum hippocampal tissue was obtained from cognitively normal HIV- and HIV+ patients, patients diagnosed with either mild cognitive/motor disorder (MCMD) or HIV-associated dementia (HAD). Immunohistochemistry and quantitative real-time PCR (qRT-PCR) for ERalpha and glial fibrillary acidic protein (GFAP) showed that ERalpha mRNA levels were not significantly different between groups, while GFAP increased in the hippocampus in the HIV+ compared to the HIV- group and was decreased in the MCMD and HAD subgroups compared to HIV+ controls. Notably the ratio of ERalpha-positive reactive astrocytes to total reactive astrocytes increased and significantly correlated with the severity of cognitive impairment following HIV infection. The data suggest that E2 would have the most dramatic effect in reducing HIV transcription early in the disease process when the subpopulation of astrocytes expressing ERalpha is low.

A novel antiestrogen agent Shikonin inhibits estrogen-dependent gene transcription in human breast cancer cells

Shikonin (SK) has been isolated and identified as a key bioactive component in an herbal plant, Shikon (gromwell). In this study, we investigated antiestrogen activity of SK in breast cancer cells. In human breast cancer cells, we observed that treatment with SK inhibits tumor cell growth in estrogen receptor alpha (ERalpha)-positive, but not ERalpha-negative breast cancer cells. Estrogen-dependent cell growth was inhibited by co-treatment with SK. A potential molecular mechanism by which SK inhibits estrogen action was explored. We found that SK has no effect on ERalpha mRNA expression, but decreases its protein level. This effect is associated with an increase in ubiquitinated ERalpha for degradation. Our results suggest that SK downregulates ERalpha protein through a proteasome-mediated pathway. We also found that the treatment with SK inhibits estrogen-induced estrogen response elements reporter gene activity. Furthermore, SK inhibits recruitment of ERalpha at the estrogen-dependent gene promoters, and subsequently suppresses gene expression. Finally, co-treatment with SK enhanced sensitivity of breast cancer cells to endocrine therapy. Collectively, our studies suggested that SK has a potential for antihormone therapy in ERalpha-positive breast cancer cells, and should serve as a target for new drug developments.

Nuclear Factor-κB Regulates Estrogen Receptor-α Transcription in the Human Heart

Estrogen receptor (ER)-mediated effects have been associated with the modulation of myocardial hypertrophy in animal models and in humans, but the regulation of ER expression in the human heart has not yet been analyzed. In various cell lines and tissues, multiple human estrogen receptor alpha (hERalpha) mRNA isoforms are transcribed from distinct promoters and differ in their 5'-untranslated regions. Using PCR-based strategies, we show that in the human heart the ERalpha mRNA is transcribed from multiple promoters, namely, A, B, C, and F, of which the F-promoter is most frequently used variant. Transient transfection reporter assays in a human cardiac myocyte cell line (AC16) with F-promoter deletion constructs demonstrated a negative regulatory region within this promoter. Site-directed mutagenesis and electrophoretic mobility shift assays indicated that NF-kappaB binds to this region. An inhibition of NF-kappaB activity by parthenolide significantly increased the transcriptional activity of the F-promoter. Increasing NF-kappaB expression by tumor necrosis factor-alpha reduced the expression of ERalpha, indicating that the NF-kappaB pathway inhibits expression of ERalpha in human cardiomyocytes. Finally, 17beta-estradiol induced the transcriptional activity of hERalpha promoters A, B, C, and F. In conclusion, inflammatory stimuli suppress hERalpha expression via activation and subsequent binding of NF-kappaB to the ERalpha F-promoter, and 17beta-estradiol/hERalpha may antagonize the inhibitory effect of NF-kappaB. This suggests interplay between estrogen/estrogen receptors and the pro-hypertrophic and inflammatory responses to NF-kappaB.

An Evaluation of Estrogenic Activity of Parabens Using Uterine Calbindin-D9k Gene in an Immature Rat Model

In the present study, calbindin-D9k (CaBP-9k), a potent biomarker for screening estrogen-like environmental chemicals in vivo and in vitro, was adopted to examine the potential estrogen-like property of the following parabens: propyl-, isopropyl-, butyl-, and isobutylparaben. Immature female rats were administered for 3 days from postnatal day 14 to 16 with 17alpha-ethinylestradiol (EE, 1 mg/kg body weight [BW]/day) or parabens (62.5, 250, and 1000 mg/kg BW/day). In uterotrophic assays, significantly increased uterus weights were detected in the EE-treated group and in the groups treated with the highest dose of isopropyl-, butyl-, and isobutylparaben. In addition, these parabens induced uterine CaBP-9k messenger RNA (mRNA) and protein levels, whereas cotreatment of parabens and fulvestrant, a pure estrogen receptor (ER) antagonist, completely reversed the paraben-induced gene expression and increased uterine weights. To investigate the ER-mediated mechanism(s) by which parabens exert their effects, the expression level of ER-alpha and progesterone receptor (PR) was analyzed. Exposure to EE or parabens caused a dramatic decrease in expression of both ER-alpha mRNA and protein levels, whereas cotreatment with fulvestrant reversed these effects. These data showed the difference of CaBP-9k and ER-alpha expression, suggesting that CaBP-9k may not express via ER-alpha pathway. In the effect of parabens on CaBP-9k expression through PR mediation, a significantly increased expression of uterine PR gene, a well-known ER-regulating gene, at both transcriptional and translational levels was indicated in the highest dose of isopropyl- and butylparaben. These parabens-induced PR gene expression was completely blocked by fulvestrant. This result indicates that CaBP-9k expression may involve with PR mediates in the estrogenic effect of paraben in immature rat uteri. Taken together, parabens exhibited an estrogen-like property in vivo, which may be mediated by a PR and/or ER-alpha signaling pathway. In addition, our results expanded the current understanding of the potential adverse effects of parabens associated with their estrogen-like activities. Further investigation is needed to elucidate in greater detail the adverse effects of parabens in humans and wildlife.

Perinatal exposure to diesel exhaust affects gene expression in mouse cerebrum

Many environmental toxins alter reproductive function and affect the central nervous system (CNS). Gonadal steroid hormones cause differentiation of neurons and affect brain function and behavior during the perinatal period, and the CNS is thought to be particularly susceptible to toxic insult during this period. It was, therefore, hypothesized that inhalation of diesel exhaust (DE) during the fetal or suckling period would disrupt the sexual differentiation of brain function in mice, and the effects of exposure to DE during the perinatal period on sexual differentiation related gene expression of the brain were investigated. In the fetal period exposure group, pregnant ICR mice were exposed to DE from 1.5 days post-coitum (dpc) until 16 dpc. In the neonatal period exposure group, dams and their offspring were exposed to DE from the day of birth [postnatal day (PND)-0] until PND-16. Then, the cerebrums of males and females at PND-2, -5, and -16 from both groups were analyzed for expression level of mRNA encoding stress-related proteins [cytochrome P450 1A1 (CYP1A1), heme oxygenase-1 (HO-1)] and steroid hormone receptors [estrogen receptor alpha (ER alpha), estrogen receptor beta (ER beta), androgen receptor (AR)]. Expression levels of ER alpha and ER beta mRNA were increased in the cerebrum of newborns in the DE exposure groups as well as mRNA for CYP1A1 and HO-1. Results indicate that perinatal exposure to DE during the critical period of sexual differentiation of the brain may affect endocrine function.

An Evaluation of Estrogenic Activity of Parabens Using Uterine Calbindin-D9k Gene in Rat Models.

Although several studies have been demonstrated the estrogenic activities of parabens, the information associated with their estrogenicity in vivo is not fully elucidated. In the present study, calbindin-D9k (CaBP-9k), a potent biomarker for screening estrogen-like environmental chemicals in vivo and in vitro, was adopted to examine the potential estrogen-like property of parabens; propyl-, isopropyl-, butyl- and isobutyl-paraben. Immature female rats were administrated for three days from postnatal day 14 to 16 with 17alpha-ethinylestradiol (EE, 1mg/kg body weight (BW)/day) and parabens (62.5, 250 and 1000 mg/kg BW/day). In uterotrophic assay, a significant increased uterus weights were detected in the EE treated groups and the highest doses of isopropyl-, butyl- and isobutyl-parabens injected rats. These parabens were further induced uterine CaBP-9k mRNA and protein levels whereas co-treatment with ICI 182,780, a pure estrogen receptors (ERs) antagonist, completely reversed the paraben-induced gene expressions and wet uterine weights. As expected, administration with parabens increased significantly in expression of uterine progesterone receptor (PR) gene, a well known ER regulating gene, at both transcriptional and translational levels. In order to bring a better understanding in ER-mediated mechanism(s) by which parabens exert their effects, the expression level of ERalpha was analyzed. Exposure to EE or parabens caused a dramatically decreased expression of the both ERalpha mRNA and protein levels but any significant difference was observed in the parabens plus ICI 182,780 treated groups. Based on these studies, it suggested that ERalpha may mediate the estrogenic effect of parabens. Taken together, parabens exhibited an estrogen-like property in vivo and it might be mediated by the ERalpha signaling pathway. In addition, these findings also offered an advanced knowledge in the adverse effects of parabens associated with their estrogen-like activities and further investigation are required to elucidate more details of adverse effects of parabens in human and wildlife. (poster)

Transcriptional Interaction of an Estrogen Receptor Splice Variant and ErbB4 Suggests Convergence in Gene Susceptibility Pathways in Schizophrenia

Mounting evidence from clinical and basic research suggests that estrogen signaling may be altered in the brains of people with schizophrenia. Previously, we found that DNA sequence variation in the estrogen receptor (ER) alpha gene, lower ERalpha mRNA levels, and/or blunted ERalpha signaling is associated with schizophrenia. In this study, we asked whether the naturally occurring truncated ERalpha isoform, Delta7, which acts as a dominant negative, can attenuate gene expression induced by the wild-type (WT) receptor in an estrogen-dependent manner in neuronal (SHSY5Y) and non-neuronal (CHOK1 and HeLa) cells. In addition, we determined the extent to which ERalpha interacts with NRG1-ErbB4, a leading schizophrenia susceptibility pathway. Reductions in the transcriptionally active form of ErbB4 comprising the intracytoplasmic domain (ErbB4-ICD) have been found in schizophrenia, and we hypothesized that ERalpha and ErbB4 may converge to control gene expression. In the present study, we show that truncated Delta7-ERalpha attenuates WT-ERalpha-driven gene expression across a wide range of estrogen concentrations in cells that express functional ERalpha at base line or upon co-transfection of full-length ERalpha. Furthermore, we find that ErbB4-ICD can potentiate the transcriptional activity of WT-ERalpha at EREs in two cell lines and that this potentiation effect is abolished by the presence of Delta7-ERalpha. Immunofluorescence microscopy revealed nuclear co-localization of WT-ERalpha, Delta7-ERalpha, and ErbB4-ICD, whereas immunoprecipitation assays showed direct interaction. Our findings demonstrate convergence between ERalpha and ErbB4-ICD in the transcriptional control of ERalpha-target gene expression and suggest that this may represent a convergent pathway that may be disrupted in schizophrenia.

Prolactin and estradiol utilize distinct mechanisms to increase serine-118 phosphorylation and decrease levels of estrogen receptor α in T47D breast cancer cells

Potential interactions between prolactin (PRL) and estradiol (E2) in breast cancer cells were explored by examining the effect of PRL on estrogen receptor (ER) serine-118 phosphorylation, ER down-regulation, and E2-stimulated cell proliferation. Both E2 and PRL resulted in prolonged ERalpha serine-118 phosphorylation, but used different signaling pathways to achieve this end. Both hormones also decreased the amount of ERalpha, but the mechanisms were different: for E2, the decrease was rapid and resulted from proteasomic degradation, whereas for PRL the decrease was slow and resulted from an effect on levels of ERalpha mRNA. PRL alone had no effect on cell number, but enhanced the increase in number in response to E2. These results are the first to demonstrate similar effects of PRL and E2 on parameters considered key to E2's effects. This suggests heretofore unrecognized and potentially important interactions between these two hormones in the natural history of breast cancer.

Regulation of Oestrogen Receptor Gene Expression: New Insights and Novel Mechanisms

17Beta-oestradiol is a pleiotropic hormone with organisational, activational and protective actions in both male and female mammals. It is responsible for numerous aspects of brain development, including sexual differentiation of the brain. The organisational effects of early oestrogen exposure are essential for long-lasting cognitive and behavioural functions. Oestradiol mediates its effects through several intracellular or membrane-associated receptor proteins. In the rodent cerebral cortex, oestrogen receptor (ER) expression, as demonstrated by hormone binding studies, is high early in postnatal life and declines precipitously as the animal approaches puberty. This decline is caused by a decreased expression of ERalpha mRNA. An understanding of the mechanisms involved in the regulation of ERalpha gene expression is critical for understanding the developmental as well as postpubertal expression of the ER. Despite recent data indicating the current hormone replacement therapies can be detrimental in older women, numerous animal studies have shown that the endogenous oestrogen, 17beta-oestradiol, is neuroprotective. Specifically, low levels of oestradiol protect the cortex from cell death caused by middle cerebral artery occlusion (MCAO). The attenuation of cell death by oestradiol in this model is mediated through an ERalpha-dependent mechanism. To this end, ERalpha expression is rapidly increased after MCAO, suggesting a return to the developmental programme of gene expression within neurones. One mechanism of suppressing gene expression is by the epigenetic modification of the promoter regions, which results in gene silencing. Of the epigenetic modifications utilised by cells, DNA methylation has been intensively studied as a mechanism by which genes are both permanently and reversibly silenced. Little is known about the mechanisms of ER gene regulation in the brain; however, in breast cancer cells, both ERalpha and ERbeta are down-regulated by promoter methylation, and subsequent binding of the methyl-CpG-binding protein, MeCP2. Data from our laboratory demonstrate that the promoters of the ERalpha gene are also methylated during development and after neuronal injury, suggesting a role of DNA methylation in regulating ER expression in the brain.

The expression of ERα, ERβ and PR in lobular carcinoma in situ of the breast determined using laser microdissection and real‐time PCR

To determine the status of oestrogen receptor (ER) subtypes (ERalpha and ERbeta) in lobular carcinoma in situ (LCIS) of the breast. Forty-seven cases of LCIS and six cases with normal breast lobules were subjected to immunohistochemistry and evaluated for ERalpha, ERbeta and progesterone receptor (PR) expression. mRNA for ERalpha, ERbeta1 and ERbeta2 were quantified in LCIS and normal lobules using laser microdissection coupled with real-time polymerase chain reaction. LCIS showed a higher level of steroid receptor protein expression than normal lobules. There was no difference in ERbeta1 gene or ERbeta protein expression between normal lobules, pure LCIS, or LCIS associated with invasive breast cancer. No significant difference in expression of either ERalpha or ERbeta was found between pure LCIS and LCIS associated with invasive cancer. However, PR was significantly lower in those cases of LCIS with associated invasive than in those without synchronous invasive disease. Increased expression of steroid receptors in LCIS suggests their possible role in the biology of LCIS and, for PR, could influence the predisposition of women diagnosed with LCIS to develop invasive breast carcinoma.

Analysis different transcriptional factors in different phenotype endometrial cancer cells

To analysis the activity of transcriptional factors in endometrial cancer cell lines with different estrogen receptor subtypes. The mRNA levels of estrogen receptor (ER) was detected by quantitative RT-PCR, and the activity of transcriptional factors was also analysed by 345-channel protein/DNA array in RL-952 (the expression status of ERalpha and ERbeta both positive), HEC-1A [ERalpha (+/-), while ERbeta negative] and HEC-1B (ERalpha and ERbeta both negative). The transcription factors of NFkappaBp65 and p38MAPK with different activity were tested by enzyme-linked immunosorbent assay (ELISA) to confirm the results of protein/DNA array. The mRNA levels of ERalpha in RL-952, HEC-1A and HEC-1B were (6780+/-282), (684+/-84) and (168+/-38) copy/ng, respectively. Among 345 candidate transcriptional factors, there were 28 factors associated with ER status. Compared with RL-952 cells, 13 transcriptional activity factors were concomitantly up-regulation, while 15 concomitantly down-regulation in HEC-1A and HEC-1B cells. Transcriptional activities of TTF (1)-1, NRF-1, TCE were significantly correlated with the high-expression status of ERalpha mRNA (r=0.523, P=0.037), while RFX123 and Ikaros were significantly correlated with the low-expression status of ERalpha mRNA (r=-0.312, P=0.041). Transcriptional factors of TTF (1)-1, NRF-1, TCE may be associated with ER-mediated signal pathway, while RFX123 and Ikaros may be associated with non ER-mediated signal pathway in endometrial cancer.

Correlation of alternative splicing of the D2 dopamine receptor mRNA and estrogen receptor mRNA in the prolactinomas and gonadotrope tumors

Estradiol (E2) acts to modulate the ratio of two dopamine D2 receptor isoforms (D2L/D2S) by the nuclear estrogen receptor (ER) and to reduce dopamine's inhibitory action on PRL secretion. Here we demonstrate the correlation between the expression of ER mRNA and D2R mRNA isoforms in pituitary neoplasms cells. Twenty-four human pituitary adenomas (14 prolactinomas and 10 gonadotrope tumors) were examined for the expression of both ER mRNA and D2R mRNA by means of semi-quantitative RT-PCR analysis. No significant difference was found in ERbeta mRNA expression levels between prolactinomas and gonadotrope tumors (P = 0.871), but there was a significant difference in the expression of ERalpha mRNA (P = 0.003). The significant difference was found between the two pituitary adenomas types in both levels of D2S and D2L mRNA expression (P = 0.036 and 0.007 respectively). Furthermore, both levels of expression in prolactinomas were significantly higher than that in gonadotrope tumors. Additionally, a negative correlation between D2S and ERalpha mRNA expression and a positive correlation between D2L and ERalpha mRNA expression were found in these tumors. This study for the first time shows a good correlation between expression of ER and D2R isoforms in prolactinomas and gonadotrope tumors. Reducing the amount of the ERalpha in neoplasm cells can alter the ratio of D2L/D2S, which may increase the drug sensitivity of pituitary adenomas.

C-fos gene and protein expression in pelvic endometriosis: a local marker of estrogen action

Endometriosis is an estrogen-dependent disease, causing pelvic pain and infertility. c-fos is an early transcription factor that has been reported to be related to estradiol-dependent cell proliferation. The aim of the present study was to assess the c-fos gene and protein expression in pelvic endometriotic implants in comparison to normal endometrium from infertile women. An open, prospective and controlled study included 15 infertile women with endometriosis and 19 control infertile women. Endometrial and endometriotic biopsies were performed at the follicular phase and the samples were processed for RT-PCR and immunohistochemistry. ERalpha mRNA levels were similar in the endometriotic implants/eutopic endometrium from women with endometriosis and in normal tissue (P = 0.649). The aromatase gene, however, was not expressed in the eutopic endometrium from either control or endometriosis groups, and was only expressed in 50% of endometriotic implants (P = 0.044). c-fos gene expression was higher in endometriotic implants (1.32 +/- 0.13; P = 0.011) than in eutopic endometrium from patients with endometriosis (0.97 +/- 0.11) or from the control group (0.91 +/- 0.05). In addition, immunohistochemistry showed a more abundant distribution of c-Fos in the stroma of endometriotic tissue compared to eutopic endometrium. These data suggest that c-fos may play a role in the molecular mechanisms of estrogen action on the induction, promotion or progression of endometriosis.

Isolation of Novel Isoforms of Estrogen Receptor Genes from Xenopus Gonad and Brain

We isolated four variants of ER alpha mRNA, two each from the brain and gonad of the Xenopus embryo (brain, bER alpha 1 and bER alpha 2; gonad, gER alpha 1 and gER alpha 2). The N-terminal-domain of ER alpha differed between the brain and gonad. While the C-terminal region downstream of the DNA-binding domain of ER alpha differed between ER alpha 1 and ER alpha 2 in both the brain and gonad, each of ER alpha 1 and ER alpha 2 contained the same sequence sets (bER alpha 1 and gER alpha 1; bER alpha 2 and gER alpha 2) between the brain and gonad. Both bER alpha 1 and gER alpha 1 contained the same full-length, C-terminal estrogen receptor, whereas the C terminus of bER alpha 2 and gER alpha 2 lacked half of the DNA-binding, ligand-binding, and transcriptional activation domain. Although gER alpha was mainly expressed in the gonad, both bER alpha and gER alpha were expressed in the brain of the Xenopus embryo at stage 50. These isoforms might be transcribed by alterative splicing of a single gene. Two gonad ER beta s differing in the length of the N-terminal sequence were isolated from the Xenopus gonad. While ER beta was expressed in the gonad, it was not expressed in the brain of the Xenopus embryo at stage 50.

Estrogen Receptor (ER) β Regulates ERα Expression in Stromal Cells Derived from Ovarian Endometriosis

Estradiol and its nuclear receptors, estrogen receptor (ER) alpha and ERbeta, play critical roles in endometrium and endometriosis. Levels of ERbeta, due to pathological hypomethylation of its promoter, are significantly higher in endometriotic vs. endometrial tissue and stromal cells, whereas ERalpha levels are lower in endometriosis. Estradiol regulates ERalpha gene expression via its alternatively used promoters A, B, and C. The aim of the study was to determine whether high levels of ERbeta in endometriotic stromal cells from ovarian endometriomas regulate ERalpha gene expression. ERbeta knockdown significantly increased ERalpha mRNA and protein levels in endometriotic stromal cells. Conversely, ERbeta overexpression in endometrial stromal cells decreased ERalpha mRNA and protein levels. ERbeta knockdown significantly decreased proliferation of endometriotic stromal cells. Chromatin immunoprecipitation assays demonstrated that estradiol enhanced ERbeta binding to nonclassical activator protein 1 and specificity protein 1 motifs in the ERalpha gene promoters A and C and a classic estrogen response element in promoter B in endometriotic stromal cells. High levels of ERbeta suppress ERalpha expression and response to estradiol in endometrial and endometriotic stromal cells via binding to classic and nonclassic DNA motifs in alternatively used ERalpha promoters. ERbeta also regulates cell cycle progression and might contribute to proliferation of endometriotic stromal cells. We speculate that a significantly increased ratio of ERbeta:ERalpha in endometriotic tissues may also suppress progesterone receptor expression and contribute to progesterone resistance. Thus, ERbeta may serve as a significant therapeutic target for endometriosis.

Role of Estrogen Receptor‐α and −β in Regulating Leptin Expression in 3T3‐L1 Adipocytes

We investigated the effects of the estrogen receptor-alpha (ERalpha) and -beta (ERbeta) in the regulation of leptin, resistin, and adiponectin expression in 3T3-L1 adipocytes. Mature adipocytes were exposed to estradiol (E2), ERalpha agonist (PPT (4,4',4''-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol)), ERbeta agonist (DPN (2,3-bis(4-Hydroxyphenyl)-propionitrile)), E2 with ERalpha antagonist (MPP (1,3-Bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole dihydrochloride)), and E2 with ERbeta antagonist (R,R-THC ((R,R)-5,11-diethyl-5,6,11,12-tetrahydro-2,8-chrysenediol)) at different concentrations. To clarify the expression and regulation of adipokines by ER subtypes, total RNA was extracted from cells and measured using quantitative PCR. Western blot analysis was performed to evaluate the protein expression of adipokines, ERalpha, and ERbeta. The leptin expression was significantly increased in the cells treated with high concentrations (10(-5) and 10(-6) mol/l) of the PPT (P < 0.01, P < 0.05). By contrast, the leptin expression decreased in a dose-dependent manner in the MPP-treated groups (P < 0.05). High concentrations (10(-5) mol/l) of R,R-THC with E2 (10(-7) mol/l) caused a significant increase of the leptin expression (P < 0.01). The leptin mRNA levels were positively correlated with the ERalpha mRNA levels (r = 0.584, P < 0.01) and negatively correlated with the ERbeta mRNA levels (r = -0.236, P = 0.03) in the adipocytes. The ratio of the ERalpha to ERbeta mRNA levels in the adipocytes was significantly associated with leptin mRNA levels (r = 0.454, P < 0.01). ERalpha induced leptin expression and ERbeta inhibited its expression in 3T3-L1 adipocytes. The ratio of the ERalpha-to-ERbeta expression in 3T3-L1 adipocytes may be an important potential regulatory factor in leptin expression.

Evidence for the expression of estrogen receptors in osteogenic cells isolated from hen medullary bone

Medullary bone is a unique tissue in female birds and forms in the cavity of long bones. This bone displays rapid remodeling in response to circulating estrogen levels, suggesting that the osteoblasts in this bone are highly sensitive to estrogen. The present study examined expression of two estrogen receptor (ER) mRNAs in osteogenic cells of medullary bone of white Leghorn hens in vitro. At day 3, isolated cells from the hen medullary bone expressed alkaline phosphatase activity. Using immunocytochemistry, ER protein was demonstrated in the nuclei of these cells. RT-PCR analysis revealed that ER-alpha mRNA was constantly expressed from day 3 to day 15 of culture, while ER-beta mRNA was not detected throughout the culture period. These results indicate that estrogen may act via ER-alpha, but not ER-beta, on osteogenic cells of the avian medullary bone.