Maternal estrogen receptor-beta expression during mouse gestation

PROBLEM: Although estrogen receptor (ER)-alpha has been well characterized, the recently identified novel ER-beta has not. In some tissues, there is overlap of the ERs, which allows for rescue in cases of deficiency; in other tissues, the ERs appear to have opposite effects. The objective of this study was to evaluate the expression of ER-beta during pregnancy. METHOD OF STUDY: Pregnant mouse uteri (embryonic days 6-14, 16, 18) were studied. ER-alpha and ER-beta oligonucleotide probes were end-labeled and in situ hybridization histochemistry was performed. RESULTS: ER-beta was strongly expressed in maternal ovaries; there was no other evidence of strong expression during gestation. ER-alpha was expressed in the uterus throughout gestation, with decreasing intensity as gestation progressed, and in maternal ovarian tissue. CONCLUSIONS: Differential expression of the two ERs was apparent during pregnancy, with ER-alpha playing a dominant role. This may have implications for selective drug treatment targeting estrogen receptors.     

Expression of oestrogen receptor-alpha and -beta in ovarian endometriomata.

The contribution of oestrogen receptor (ER) isoforms, ER-alpha and ER-beta, in oestrogen-dependent development and growth of ovarian endometriomata, is unknown. Therefore, we examined the expression of ER-alpha and ER-beta in ovarian endometriomata and normal uterine endometrium. ER-alpha and ER-beta were shown to be dominantly expressed in the nuclei of the epithelial lining cells of ovarian endometrioma and of the glandular cells of normal uterine endometrium. ER-beta was expressed at a much lower level than ER-alpha in the glandular cells of normal uterine endometrium, while ER-beta was expressed at a slightly lower level than ER-alpha in the epithelial lining cells of ovarian endometrioma. In normal uterine endometrium, ER-beta mRNA was expressed at a much lower level than ER-alpha mRNA, and the expression pattern of ER-beta mRNA during the menstrual cycle was similar to that of ER-alpha mRNA. On the other hand, ER-beta mRNA expression was significantly higher and over a much greater range in ovarian endometriomata (P < 0.05) than in normal uterine endometrium during the menstrual cycle, while ER-alpha mRNA expression was relatively lower and more random. Therefore, in ovarian endometriomata, oestrogen action via ER-alpha cascades seems to be partially damaged, as the expression of ER-alpha mRNA does not respond to endocrinological alterations during the menstrual cycle, while the relative over-expression of ER-beta might be related to a unique oestrogen-dependent growth and spreading of ovarian endometriomata.     

Estrogen receptor beta expression in invasive breast cancer

The aim of this work was to determine the extent of estrogen receptor beta (ER-beta) expression in invasive breast cancer (BrCA) and whether ER-beta expression is correlated with response to adjuvant hormonal therapy with tamoxifen (AHTT). Immunohistochemical staining (IHC) for estrogen receptor alpha (ER-alpha) and ER-beta was performed on sections of formalin-fixed and paraffin-embedded tissue from 47 unselected invasive breast carcinomas (BrCA). IHC for ER-beta was also performed on sections of BrCA from 118 women who were treated with mastectomy and AHTT. Survival analysis was performed using the Kaplan-Meier method and the log-rank test. Of the 47 unselected BrCA, 17 (36%) were negative for ER-alpha and of these, 8 (47% of ER-alpha negative cases and 17% of all 47 patients) were ER-beta positive. Five of the 8 ER-alpha negative and ER-beta positive cases were positive for ER biochemically. There was no correlation between ER-beta positivity and overall survival in the unselected group. By contrast, in the group of women treated with AHTT, expression of ER-beta in more than 10% of cancer cells was associated with better survival (P = .0077), even in women with node-negative BrCA (P = .0069). In conclusion, our results show that a significant number of women with BrCA are positive for ER-beta only, and may be determined to be ER-negative when currently available IHC is used. ER-beta status is a significant predictor of response to AHTT in women with BrCA. Larger studies with multivariate analysis are needed to confirm these findings.     

Effects of testosterone and estrogen treatment on the distribution of sex hormone receptors in the endometrium of postmenopausal women

OBJECTIVE: Our aim was to investigate the effects of the addition of testosterone to estrogen compared with those of estrogen alone on the expression and distribution of sex hormone receptors in glands and stroma of the endometrium of postmenopausal women. DESIGN: An open, randomized clinical study with parallel group comparison was performed in the Women's Health Research Unit at a university hospital. Thirty-one postmenopausal women were given oral estradiol valerate (2 mg daily) or estradiol valerate in combination with testosterone undecanoate (40 mg every 2 days) for 3 months. Before and at the end of treatment, endometrial biopsy samples were obtained, and expressions of estrogen receptor (ER)-alpha, ER-beta, progesterone receptor isoforms A and B, and androgen receptor (AR) were evaluated by immunohistochemical analysis. RESULTS: At baseline, expressions of ER-alpha and progesterone receptors were stronger in glands than in stroma, whereas the immunostaining of AR was stronger in stroma than in glands. After treatment, expressions of ER-alpha and progesterone receptors were up-regulated in both glands and stroma by both treatments, but to a lesser extent in glands by combined treatment. The expression of ER-beta in glands was significantly higher with combined treatment than with estrogen alone. Moreover, AR immunostaining was significantly higher after combined treatment than after treatment with estrogen alone. CONCLUSIONS: Expressions of AR and ER-beta were stronger in glands of the endometrium of postmenopausal women after treatment with testosterone added to estrogen than after estrogen alone. In contrast, expressions of ER-alpha and progesterone receptors were up-regulated in the endometrium with estrogen-alone treatment, whereas these expressions were less increased in glands after combined treatment. These data indicate that testosterone is involved in the regulation of sex hormone receptor expression in the postmenopausal endometrium and may therefore influence endometrial proliferation and differentiation.     

Presynaptic kainate receptors play different physiological roles in mossy fiber and associational-commissural synapses in CA3 of hippocampus from adult rats

Immunoreactivities for estrogen receptor-alpha (ER-alpha) and ER-beta are expressed in sensory neurons of the dorsal root ganglia (DRG). It has not been established, however, if the two receptor subtypes coexist in the same neuron. Double-staining immunohistochemical techniques were used to determine if subpopulations of neurons in the lumbosacral DRG exist based on their content of ERs. Results indicate that some neurons (approximately 17%) of the L6-S1 DRG contain ER-alpha -, some (approximately 23%) contain ER-beta - immunoreactivity and some (approximately 5%) express immunoreactivity for both subtypes of the ER. These results suggest that many sensory neurons can respond to estrogens, but estrogens may produce different morphofunctional effects in different neurons based on their expression of ER subtypes.     

Direct involvement of breast tumor fibroblasts in the modulation of tamoxifen sensitivity

Using contact-dependent three-dimensional coculture systems and serum-free conditions, we compared the ability of estrogen receptor (ER)-alpha(+) tamoxifen-sensitive premalignant (EIII8) or tumorigenic (MCF-7), ER-alpha(+) tamoxifen-resistant (EIII8-TAM(R)) or ER-alpha(-) MDA-MB-231 breast cancer cells to interact and undergo epithelial morphogenesis on association with breast tumor-derived fibroblasts. Although all breast cancer cell lines interacted with tumor fibroblasts, EIII8 and its intrinsically tamoxifen-resistant counterpart EIII8-TAM(R) cells were most receptive and responded with dramatic, albeit, aberrant epithelial morphogenesis. EIII8 cells underwent epithelial morphogenesis when cocultured with fibroblasts from ER-alpha(-)/PgR(-) or ER-alpha(+)/PgR(+) breast tumors; however, EIII8 cells cocultured with ER-alpha(-)/PgR(-) tumor-derived fibroblasts exhibited decreased tamoxifen sensitivity compared with cells cocultured with ER-alpha(+)/PgR(+) tumor fibroblasts. Fibroblast-induced tamoxifen resistance was accompanied by mitogen-activated protein kinase and Akt hyperactivation, reduced sensitivity to U0126 or LY294002, and ER-alpha hyperphosphorylation in the activation function-1 domain. The intrinsic tamoxifen resistance of EIII8-Tam(R) cells correlated with constitutive ER-alpha hyperphosphorylation that was unaffected by the tumor fibroblasts. Our results suggest that tumor fibroblast-induced tamoxifen resistance of EIII8 cells is not mediated by epidermal growth factor receptor or insulin-like growth factor (IGF)-1R axes because no correlation was found between expression levels of IGF-1, IGF-2, phosphorylated IGF-1R, or epidermal growth factor receptor, and tamoxifen sensitivity of EIII8 fibroblast cultures.     

Differential distribution of estrogen receptor (ER)-alpha and ER-beta in the midbrain raphe nuclei and periaqueductal gray in male mouse: Predominant role of ER-beta in midbrain serotonergic systems

We examined the distribution of estrogen receptor (ER)-alpha and ER-beta immunoreactive (ir) cells in the dorsal (DRN) and median/paramedian (MPRN) raphe nuclei in male mice. ER-alpha ir neurons were scattered across the three subdivisions (ventral, dorsal, and lateral) of the DRN and the MPRN. Robust ER-beta ir cells were observed throughout the raphe nuclei, and were particularly abundant in the ventral and dorsal subdivisions of the DRN. Using dual-label immunocytochemistry for ER-alpha or ER-beta with tryptophan hydroxylase (TPH), the rate-limiting enzyme for 5-hydroxytryptamine (5-HT) synthesis, over 90% of ER-beta ir cells exhibited TPH-ir in all DRN subdivisions, whereas only 23% of ER-alpha ir cells contained TPH. Comparisons of ER-alpha knockout (alphaERKO) as well as ER-beta knockout (betaERKO) mice with their respective wild-type (WT) littermates revealed that gene disruption of either ER-alpha or ER-beta did not affect the other ER subtype expression in the raphe nuclei. In situ hybridization histochemistry revealed that there was a small but statistically significant decrease in TPH mRNA expression in the ventral DRN subdivision in betaERKO mice compared with betaWT mice, whereas TPH mRNA levels were not affected in alphaERKO mice. These findings support a hypothesis that ER-beta activation may contribute to the estrogenic regulation of neuroendocrine and behavioral functions, in part, by acting directly on 5-HT neurons in the raphe nuclei in male mice.     

Estrogen specifically stimulates expression and production of osteoprotegerin from rheumatoid synovial fibroblasts

We studied the effects of estrogen on human fibroblast-like synovial cells in rheumatoid arthritis (RA-FLS) focusing on receptor activator of NF-kappaB ligand (RANKL) and its decoy receptor osteoprotegerin (OPG), the osteoclast formation and function regulators that have a substantial role in bone erosion of RA. Estrogen influences osteoporosis and the onset of RA clinically. The cellular responses of RA-FLS to estrogen are initiated via two high-affinity estrogen receptors (ERs). Culture of RA-FLS in the presence of 10(-6) M 17beta-estradiol (E2) increased expression of estrogen receptor (ER)-alpha, but not ER-beta. OPG mRNA expression was significantly increased, whereas RANKL mRNA was unaffected. E2 treatment also significantly increased the amount of OPG released in the culture supernatant. The increase of OPG and ER-alpha was specifically antagonized by the pure estrogen antagonist ICI 182780. Tamoxifen, a selective ER moderator, did not increase OPG. The results indicate that estrogen stimulates secretion of OPG from RA-FLS by acting on ER-alpha, which likely prevents bone erosion in RA.     

Expression of oestrogen receptor-beta in apocrine carcinomas of the breast

AIMS: Apocrine carcinoma of the breast seldom expresses oestrogen receptors (ER) or progesterone receptors (PR), but frequently expresses androgen receptors (AR). Because of this unusual hormone receptor status, it has been suggested that oestrogens have a less important role in the pathogenesis of apocrine carcinoma. The ER status of apocrine carcinoma has been studied for one kind of ER, the classic receptor now named ER-alpha; however, the status of ER-beta, a secondary oestrogen receptor, has not been examined systematically in apocrine carcinoma. The aim was to study ER-beta status in apocrine carcinoma. METHODS AND RESULTS: The expression of ER-beta was examined immunohistochemically in 48 apocrine carcinomas and compared with clinicopathological factors and ER-alpha, PR and AR status. ER-beta positivity was observed in 35 cases (73%), regardless of any clinicopathological factors or the status of other receptors. The results of ER-beta mRNA analysis supported the immunohistochemical results. CONCLUSIONS: The significance of oestrogens in apocrine carcinoma should not be dismissed at present when the role of ER-beta remains to be determined. Studying the action of oestrogen or antioestrogen in apocrine carcinoma may reveal a role for ER-beta independent of ER-alpha and raise the potential of hormonal therapy for these tumours.     

Tissue-specific expression of estrogen receptors and their role in the regulation of neutrophil infiltration in various organs following trauma-hemorrhage

Although 17beta-estradiol (E2) administration after trauma-hemorrhage (T-H) reduces tissue neutrophil sequestration in male rodents, it remains unknown which of the estrogen receptor (ER) subtypes mediates this effect and whether the same ER subtype is involved in all the tissues. We hypothesized that the salutary effects of E2 on attenuation of neutrophil accumulation following T-H are tissue and receptor subtype-specific. Male Sprague-Dawley rats underwent sham operation or T-H (mean blood pressure, 40 mmHg for 90 min and then resuscitation). E2 (50 microg/kg), ER-alpha agonist propyl pyrazole triol (PPT; 5 microg/kg), ER-beta agonist diarylpropiolnitrile (DPN; 5 microg/kg), or vehicle (10% dimethyl sulfoxide) was administered subcutaneously during resuscitation. Twenty-four hours thereafter, tissue myeloperoxidase (MPO) activity (a marker of neutrophil sequestration), cytokine-induced neutrophil chemoattractant (CINC)-1, CINC-3, and intercellular adhesion molecule (ICAM)-1 levels in the liver, intestine, and lung were measured (n = 6 rats/group). ER-alpha and ER-beta mRNA levels in sham-operated rats were also determined. T-H increased MPO activity, CINC-1, CINC-3, and ICAM-1 levels in the liver, intestine, and lung. These parameters were improved significantly in rats receiving E2 after T-H. Administration of the ER-alpha agonist PPT but not the ER-beta agonist DPN improved the measured parameters in the liver. In contrast, DPN but not PPT significantly improved these parameters in the lung. In the intestine, ER subtype specificity was not observed. ER-alpha mRNA expression was highest in the liver, whereas ER-beta mRNA expression was greatest in the lung. Thus, the salutary effects of E2 administration on tissue neutrophil sequestration following T-H are receptor subtype and tissue-specific.     

The expression of estrogen receptors in hepatocellular carcinoma in Korean patients

Expression of estrogen receptors (ER)-alpha and -beta, as well as androgen receptor (AR), in hepatocellular carcinoma (HCC) is thought to be correlated with prognosis, survival, and male prevalence of HCC. These hypotheses are based on investigations of European patients; however the expression patterns of these receptors in Asian patients are largely unknown. In this study, we collected liver carcinoma and peritumor tissues from 32 patients (9 females and 23 males) in South Korea. The expression of ERs and ARs was studied using RT-PCR. Wild-type ER-alpha and AR were expressed in all of the samples investigated, and their expression was independent of the causal virus or patient sex. Expression of the ER-alpha variant was independent of sex (100% female vs. 91.3% male) and HCV and HBV status (91.3% vs. 100%). Wild-type ER-beta was expressed more often in HCV patients than in HBV patients (95.7% vs. 44.4%; p < 0.05). In conclusion, the stronger ER-alpha variant expression in HCC tissues implies that this variant has an important role in HCC development. However, at least in Korean patients, expression of the ER-alpha variant (vER-alpha) is not related to male HCC prevalence. In addition, the predominant expression of ER-beta in HCV patients suggests that it plays an important role in HCV-induced liver disease.     

Evidence for cell-specific changes with age in expression of oestrogen receptor (ER) alpha and beta in bone fractures from men and women

Oestrogen is recognized as important for maintaining bone mass in men and women. Oestrogen receptor (ER) alpha and the recently described ER-beta are both expressed in bone cells, but have different affinities for oestrogen agonists and plant oestrogens, which could be important in developing treatments for bone loss in both men and women. It is unclear, however, which isoform predominates in bone; cell type and age may influence their relative expression. The present study has compared ER-alpha and ER-beta expression in serial sections of human fracture callus from males (n = 19, age range 5-72 years) and females (n = 15, age range 3-86 years) by indirect immunoperoxidase. Fracture callus was used as it can be readily obtained from individuals over a wide age range and contains a variety of bone cells. Antibody specificity was confirmed by western blotting and comparison of immunoreactivity in sections of breast tumour and benign prostate hyperplasia. No gender difference in ER expression was found in bone from individuals less than 40 years old. Proliferative chondrocytes were positive for both isoforms, but few larger hypertrophic cells were immunoreactive. ER-alpha and ER-beta were co-expressed in osteoclasts, suggesting that oestrogen may act directly on these cells. Osteoblasts, osteocytes, and mesenchymal cells also expressed both isoforms. In women over 40 years of age, however, relatively fewer biopsies contained osteocytes positive for ER-alpha and ER-beta. Likewise, the proportions of osteoblasts and mesenchymal cells expressing ER-beta were reduced but ER-alpha remained unaffected. In contrast, in men over 40 years, only the proportion of biopsies containing ER-beta-positive mesenchymal cells was lower. In these older men and women, ER-alpha and ER-beta expression was retained by the small proliferative chondrocytes. These results demonstrate that gender, age, and cell type are important determinants of ER isoform expression in skeletal cells.