AB1410 Sigma-AldrichAnti-Estrogen Receptor β Antibody, human ER-β

Estrogen has been reported to accelerate cutaneous wound healing. This research studies the effect of young coconut juice (YCJ), presumably containing estrogen-like substances, on cutaneous wound healing in ovairectomized rats.Four groups of female rats (6 in each group) were included in this study. These included sham-operated, ovariectomized (ovx), ovx receiving estradiol benzoate (EB) injections intraperitoneally, and ovx receiving YCJ orally. Two equidistant 1-cm full-thickness skin incisional wounds were made two weeks after ovariectomy. The rats were sacrificed at the end of the third and the fourth week of the study, and their serum estradiol (E2) level was measured by chemiluminescent immunoassay. The skin was excised and examined in histological sections stained with H&E, and immunostained using anti-estrogen receptor (ER-α an ER-β) antibodies.Wound healing was accelerated in ovx rats receiving YCJ, as compared to controls. This was associated with significantly higher density of immunostaining for ER-α an ER-β in keratinocytes, fibroblasts, white blood cells, fat cells, sebaceous gland, skeletal muscles, and hair shafts and follicles. This was also associated with thicker epidermis and dermis, but with thinner hypodermis. In addition, the number and size of immunoreactive hair follicles for both ER-α and ER-β were the highest in the ovx+YCJ group, as compared to the ovx+EB group.This study demonstrates that YCJ has estrogen-like characteristics, which in turn seem to have beneficial effects on cutaneous wound healing.

The estrogen receptor (ER) signaling and the insulin-like growth factor-1 receptor (IGF-1R) signaling are implicated in lung cancer progression. Here, we sought to investigate whether estrogen regulated the IGF-1R signaling in non-small cell lung cancer (NSCLC) and the underlying mechanisms. We examined and analyzed the correlation of the expression of aromatase (Arom), ERβ, ERα, insulin-like growth factor-1 (IGF-1), and IGF-1R in NSCLC. Tissue-microarray and immunohistochemistry analysis of tissue specimens from 162 NSCLC patients and 38 patients with benign pulmonary lesions showed that Arom, ERβ, IGF-1, and IGF-1R were overexpressed while ERα was not expressed in NSCLC. Furthermore, ERβ expression was positively correlated with that of Arom, IGF-1, and IGF-1R (r = 0.554, 0.649, 0.496, respectively, P values are equal to 0.000), while Arom expression was positively associated with that of IGF-1 and IGF-1R (r = 0.657, 0.714, respectively, P values are equal to 0.000). Additionally, ERβ, IGF-1, and phospho-IGF-1R, but not ERα, were expressed in A549 cells. Immunoblotting assays showed that A549 cells treated with E2 showed significantly higher IGF-1 and p-IGF-1R levels than those receiving the combination treatment of 17β-estradiol (E2) and fulvestrant (Ful, ER antagonist) (P = 0.042, 0.002, respectively) or controls (P values are equal to 0.000). The MTT assays further revealed that E2 and IGF-1 synergistically promoted A549 cell proliferation. Together, our study provides the first direct evidence for an interaction between ER and IGF-1R in lung cancer. We showed that estrogen upregulated the IGF-1R signaling through ERβ in lung cancer tissues and A549 cells. These findings shed further light on the mechanisms whereby estrogen promotes lung cancer and highlight the ER and IGF-1R signaling pathways as promising targets for combinational therapy for lung cancer.

The role of estrogen receptor alpha (ERα) in breast cancer has been studied extensively, and its protein expression is prognostic and a primary determinant of endocrine sensitivity. However, much less is known about the role of ERβ and its relevance remains unclear due to the publication of conflicting reports. Here, we provide evidence that much of this controversy may be explained by variability in antibody sensitivity and specificity and describe the development, characterization, and potential applications of a novel monoclonal antibody targeting full-length human ERβ and its splice variant forms. Specifically, we demonstrate that a number of commercially available ERβ antibodies are insensitive for ERβ and exhibit significant cross-reaction with ERα. However, our newly developed MC10 ERβ antibody is shown to be highly specific and sensitive for detection of full-length ERβ and its variant forms. Strong and variable staining patterns for endogenous levels of ERβ protein were detected in normal human tissues and breast tumors using the MC10 antibody. Importantly, ERβ was shown to be expressed in a limited cohort of both ERα positive and ERα negative breast tumors. Taken together, these data demonstrate that the use of poorly validated ERβ antibodies is likely to explain much of the controversy in the field with regard to the biological relevance of ERβ in breast cancer. The use of the MC10 antibody, in combination with highly specific antibodies targeting only full-length ERβ, is likely to provide additional discriminatory features in breast cancers that may be useful in predicting response to therapy.

BACKGROUND: Nebivolol (NEB) is a [beta]1-receptor blocker with nitric oxide-dependent vasodilating properties. NEB-induced nitric oxide release is mediated through the estrogen receptor. METHOD: Here, we tested the hypothesis that NEB decreases endothelial cell stiffness and that these effects can be abolished by both endothelial nitric oxide synthase and estrogen receptor blockade. Human endothelial cells (EAHy-926) were incubated with vehicle, NEB 0.7 nmol/l, metoprolol 200 nmol/l, 17[beta]-estradiol (E2) 15 nmol/l, the estrogen receptor antagonists tamoxifen 100 nmol/l and ICI 182780 (ICI) 100 nmol/l, the nitric oxide synthase inhibitor N[omega]-nitro-L-arginine methyl ester 1 mmol/l and combinations of NEB and E2 with either tamoxifen, ICI or N[omega]-nitro-L-arginine methyl ester as well as metoprolol and ICI. Atomic force microscopy was performed to measure cellular stiffness, cell volume and apical surface. Presence of estrogen receptor protein in EAHy-926 was confirmed by western blot analysis; quantification of ER[alpha] and ER[beta] total RNA was performed by semiquantitative PCR. RESULTS: Both NEB as well as E2 decreased cellular stiffness to a similar extent (NEB: 0.83 +/- 0.03 pN/nm, E2: 0.87 +/- 0.03 pN/nm, vehicle: 2.19 +/- 0.07 pN/nm), whereas metoprolol had no effect on endothelial stiffness (2.07 +/- 0.04 pN/nm, all n = 60, P 0.01). The decrease in stiffness occurred as soon as 5 min after starting NEB incubation. The effects are mediated through nongenomic ER[beta] pathways, as ER[alpha] is not translated into measurable protein levels in EAHy-926. Furthermore, NEB increased cell volume by 48 +/- 4% and apical surface by 34 +/- 3%. E2 had comparable effects. Tamoxifen, ICI and N[omega]-nitro-L-arginine methyl ester substantially diminished the effects of NEB and E2. CONCLUSION: NEB decreases cellular stiffness and causes endothelial cell growth. These effects are nitric oxide-dependent and mediated through nongenomic ER[beta] pathways. The morphological and functional alterations observed in endothelial cells may explain improved endothelial function with NEB treatment.

Oestrogens are involved in regulation of spermatogenesis and sperm maturation and are essential for male fertility. To study the role of oestrogens on epididymal function in the domestic cat, we analyzed the localization patterns of oestrogen receptors (ERs) within the epididymis of juvenile, pubertal and adults using immunohistochemistry. Cat epididymal tissues obtained during routine castrations were fixed in chilled Bouin's solution and processed for immunohistochemistry with ER-specific antibodies. For a certain receptor type, ER localization was influenced by donor age. In the juvenile epididymis, ERalpha was localized in the nuclei of epithelial cells of efferent ducts and undifferentiated epithelium of the ductus epididymis. During puberty, ERalpha localization in the undifferentiated epithelium of the epididymis shifted from the nuclei to the cytoplasm and plasma membrane. Oestrogen receptor-alpha level was highest in the pubertal and adult epididymis, especially within the cytoplasm and in plasma membranes of caput epithelial cells. This finding was suggestive of a role in fluid reabsorption within the efferent ducts and the epididymis. In corpus and cauda regions, ERalpha was less abundant, suggesting a minor role for oestrogens in sperm storage areas. Interestingly, localization of ERbeta was neither influenced by age nor location within the epididymis and was ubiquitous throughout. Results demonstrate that oestrogen actions within the epididymis may be predominantly mediated through ERalpha during sexual maturation in the domestic cat.

Breast cancers (BC) in women carrying mutations in BRCA1 gene are more frequently estrogen receptor negative than the nonhereditary BC. Nevertheless, tamoxifen has been found to have a protective effect in preventing contralateral tumors in BRCA1 mutation carriers. The identification of the second human estrogen receptor, ERbeta, raised a question of its role in hereditary breast cancer. The aim of this study was to assess the frequency of ERalpha, ERbeta, PgR (progesterone receptor) and HER-2 expression in breast cancer patients with mutated BRCA1 gene and in the control group.The study group consisted of 48 women with BRCA1 gene mutations confirmed by multiplex PCR assay. The patients were tested for three most common mutations of BRCA1 affecting the Polish population (5382insC, C61G, 4153delA). Immunostaining for ERalpha, ERbeta and PgR (progesterone receptor) was performed using monoclonal antibodies against ERalpha, PgR (DakoCytomation), and polyclonal antibody against ERbeta (Chemicon). The EnVision detection system was applied. The study population comprised a control group of 120 BC operated successively during the years 1998-99.The results of our investigation showed that BRCA1 mutation carriers were more likely to have ERalpha-negative breast cancer than those in the control group. Only 14.5% of BRCA1-related cancers were ERalpha-positive compared with 57.5% in the control group (P less than 0.0001). On the contrary, the expression of ERbeta protein was observed in 42% of BRCA1-related tumors and in 55% of the control group. An interesting finding was that most hereditary cancers (75% of the whole group) were triple-negative: ERalpha(-)/PgR(-)/HER-2(-) but almost half of this group (44.4%) showed the expression of ERbeta.In the case of BRCA1-associated tumors the expression of ERbeta was significantly higher than the expression of ERalpha. This may explain the effectiveness of tamoxifen in preventing contralateral breast cancer development in BRCA1 mutation carriers.

Roe deer (a seasonal breeder, rut: July to August) is a well characterized model for studying the seasonal regulation of testicular activity. However, not much is known about the impact of estrogens on seasonally determined sperm production. We therefore explored the time and cell type specific expression of estrogen receptors and of enzymes involved in steroid biosynthesis in roe deer testicular parenchyma and in the epididymis. Every second month during the entire seasonal cycle five roe bucks were castrated (n=30). Estrogen receptor (ER) alpha, ERbeta and the enzymes P450Aromatase and P450C17 were localized immunohistochemically. The expression levels of ERalpha, ERbeta and P450Aromatase were evaluated by semi-quantitative Western blot. Contrary to the enzyme required for androgen production (P450C17), which is expectedly located only in the Leydig cells and shows an expression increase towards rutting season, a seasonal expression difference of the enzyme required for the conversion into oestradiol (P450Aromatase) is visible only in the epididymis. In the testis, ERalpha expression shows a striking dependency on tubular cell composition, and the single cell expression activity increases towards rut. This implicates that estrogens are directly involved in the regulation of spermatogenesis in the roe buck. In the epididymis, expression of ERalpha is seasonally determined particularly in the ductuli efferentes. ERbeta was detected throughout the year with no distinct dependency on season or the stages of germinative epithelium cycle. We conclude that estrogens in the roe buck influence the seasonally determined sperm production predominantly by the regulated expression of ERalpha.

AIMS: We aimed to investigate the sources of estrogen receptor alpha (ERalpha), estrogen receptor beta (ERbeta) and estimate the value of both ER subtypes in gastric adenocarcinoma and analyze the possible relationship of prothymosin alpha (ProTalpha) to ERs. METHODS: ERs at the mRNA and protein levels in matched advanced gastric adenocarcinomas and surrounding non-cancerous tissues were examined by using reverse transcription-polymerase chain reaction and immunohistochemical (IHC) methods. Cell proliferation related protein ProTalpha was also detected in IHC. The immunoreactive signal, corresponding to the proteins expression level, was quantitatively analyzed. RESULTS: Both ERalpha and ERbeta mRNAs were detected in most of the cancer and matched normal tissues analyzed. At the protein level, the percentage of ERalpha and ERbeta positive cases changed. ERalpha immunoreactivity was only detected in poorly differentiated adenocarcinoma and ERalpha positive expression correlated with depth of invasion of the tumors. Compared with non-cancerous tissues, gastric tumors showed decreased ERbeta expression and lost ERbeta. Altered ERbeta in gastric adenocarcinoma correlated with decreased differentiation. And the tumors involved lymph node metastasis showed significantly lower expression level of ERbeta. ProTalpha in ERbeta-positive tumors showed higher expression than that in lost ERbeta tumors. CONCLUSIONS: Altered expression of ERalpha and ERbeta in tumors compared with corresponding normal gastric tissues was more common in poorly differentiated adenocarcinomas and related to malignant properties, such as lymph node metastasis. Decreased ERbeta and increased ProTalpha expression in advanced gastric adenocarcinoma indicated that ERbeta may play an anti-proliferation role which is opposed to the role of ProTalpha in gastric epithelium.

The optimisation of a flow cytometric protocol for the determination of the estrogen receptor beta (ERbeta) expression in bovine blood neutrophils is described. The following final incubation conditions were obtained: fixation with 0.25% formaldehyde and 70% methanol, both for 1 h; permeabilisation with 0.05% Triton X-100, overnight labelling at 4 degrees C with the primary antibody diluted at 10 microg/ml and subsequent labelling for 30 min on ice with the fluorescein isothiocyanate-conjugated secondary antibody at 8 microg/ml. Of the three anti-human or anti-rat ERbeta primary antibodies evaluated, only PA1-311 was found to cross-react with bovine cells. Immunoblot analysis supports the obtained results. The flow cytometric technique allows reproducible quantitative determination of the ERbeta protein in neutrophils and may be a valuable tool for future expression studies in these cells of the innate immune system.

Estrogens have many cellular functions, including their interactions with estrogen receptors alpha and beta (ERalpha and ERbeta). Earlier, we determined that the estrogen-ER complex stimulates the transcriptional activity of the matrix metalloproteinase 26 (MMP-26) gene promoter. We then determined that ERbeta is susceptible to MMP-26 proteolysis whereas ERalpha is resistant to the protease. MMP-26 targets the NH(2)-terminal region of ERbeta coding for the divergent NH(2)-terminal A/B domain that is responsible for the ligand-independent transactivation function. As a result, MMP-26 proteolysis generates the COOH-terminal fragments of ERbeta. Immunohistochemical analysis of tissue microarrays derived from 121 cancer patients corroborated these data and revealed an inverse correlation between the ERalpha-dependent expression of MMP-26 and the levels of the intact ERbeta in breast carcinomas. MMP-26 is not expressed in normal mammary epithelium. The levels of MMP-26 are strongly up-regulated in ductal carcinoma in situ (DCIS). In the course of further disease progression through stages I to III, the expression of MMP-26 decreases. In contrast to many tumor-promoting MMPs, the expression of MMP-26 in DCIS correlated with a longer patient survival. Our data suggest the existence of an MMP-26-mediated intracellular pathway that targets ERbeta and that MMP-26, a novel and valuable cancer marker, contributes favorably to the survival of the ERalpha/beta-positive cohort of breast cancer patients.