Quantitative analysis of estrogen receptor proteins in rat mammary gland.

Estrogen receptor alpha and beta proteins (ERalpha and ERbeta) at various stages of development of the rat mammary gland were quantified by Western blotting. ERalpha and ERbeta recombinant proteins were used as standards, and their molar concentrations were measured by ligand binding assays. In 3-week-old pregnant, lactating, and postlactating rats the ERalpha content ranged from 0.30-1.55 fmol/microg total protein (mean values). The ERbeta content of the same samples ranged between 1.06-7.50 fmol/microg total protein. At every developmental stage, the ERbeta content of the mammary gland was higher than that of ERalpha. When receptor levels were normalized against beta-actin, it was evident that ER expression changed during development, with maximum expression of both receptors during the lactation period. With an antibody raised against the 18-amino acid insert of the ERbeta variant, originally called ERbeta2 but named ERbetains in this paper, Western blots revealed that ERbetains protein was up-regulated during the lactation period. RT-PCR showed that the levels of messenger RNA of ERbetains paralleled those of the protein. Double immunohistochemical staining with anti-ERalpha and anti-ERbetains antibodies revealed that ERbetains protein colocalized with ERalpha in 70-80% of the ERalpha-expressing epithelial cells during lactation and with 30% of these cells during pregnancy. These observations indicate that expression of ERbetains is regulated not only quantitatively, but also with regard to its cellular distribution. As ERbetains acts as the dominant repressor of ERalpha, we suggest that its coexpression with ERalpha quenches ERalpha function and may be one of the factors that contribute to the previously described insensitivity of the mammary gland to estrogens during lactation.     

Expression and localisation of oestrogen and progesterone receptors in the bovine mammary gland during development,function and involution

It is now well established that oestrogen and progesterone are absolutely essential for mammary gland development. Lactation can be induced in non-pregnant animals by sex steroid hormone treatment. Most of the genomic actions of oestrogens are mediated by two oestrogen receptors (ER)-alpha and ERbeta, and for gestagens in ruminants by the progesterone receptor (PR). Our aim was the evaluation of mRNA expression and protein (localisation and Western blotting) during mammogenesis, lactogenesis, galactopoiesis (early, middle and late) and involution (8, 24, 28, 96-108 h and 14-28 days after the end of milking) in the bovine mammary gland (total no. 53). During these stages, the mRNA was assessed by means of real-time RT-PCR (LightCycler). The protein for ERalpha, ERbeta and PR was localised by immunohistochemistry and Western blotting. The mRNA expression results indicated the existence of ERalpha, ERbeta and PR in bovine mammary gland. Both ERalpha and PR are expressed in fg/ micro g total RNA range. The highest mRNA expression was found for ERalpha and PR in the tIssue of non-pregnant heifers, followed by a significant decrease to a lower level at the time of lactogenesis with low concentrations remaining during lactation and the first 4 weeks of involution. In contrast, the expression of ERbeta was about 1000-fold lower (ag/ micro g total RNA) and showed no clear difference during the stages examined, with a significant increase only 2-4 weeks after the end of milking. Immunolocalisation for ERalpha revealed a strong positive staining in nuclei of lactocytes in non-pregnant heifers, became undetectable during pregnancy, lactogenesis and lactation, and was again detectable 14-28 days after the end of milking. In contrast, PR was localised in the nuclei of epithelial cells in the mammary tIssue of non-pregnant heifers, in primigravid animals, and during late lactation and involution. During lactogenesis, peak and mid lactation, fewer nuclei of epithelial cells were positive, but increased staining of the cytoplasm of epithelial cells was obvious. ERalpha and ERbeta protein was found in all mammary gland stages examined by Western blotting. In contrast to mRNA expression, the protein signal for ERalpha was weaker in the tIssue of non-pregnant heifers and during involution (4 weeks). ERbeta protein showed a stronger signal (two isoform bands) in non-pregnant heifers and 4 weeks after the end of milking. This correlated with the mRNA expression data. Three isoforms of PR (A, B and C) were found by Western blotting in the tIssue of non-pregnant heifers, but only isoform B remained during the following stages (lactogenesis, galactopoiesis and involution). In conclusion, the mRNA expression and protein data for ER and PR showed clear regulatory changes, suggesting involvement of these receptors in bovine mammary gland development and involution.     

Messenger ribonucleic acid in situ hybridization analysis of estrogen receptors alpha and beta in human breast carcinoma

We examined the expression of a recently characterized novel estrogen receptor (ER) beta in 25 cases of invasive ductal carcinoma of the breast, using messenger RNA (mRNA) in situ hybridization, and compared the findings with those of ERalpha, to study its localization and its possible biological significance in human breast cancer. ERalpha and ERbeta hybridization signals were both detected, predominantly in carcinoma cells and in some stromal cells, in 18 of 25 (72%) and 11 of 25 (44%) cases, respectively. The cases in which more than 25% of carcinoma cells demonstrated mRNA hybridization signals were 13 of 25 (52%) and 2 of 25 (8%) cases for ERalpha and ERbeta, respectively. Among the cases expressing ERbeta, 10 of 11 (91%) also expressed ERalpha mRNA; and in these 10 cases, coexpressing both ERalpha and beta, the number of carcinoma cells expressing ERalpha was greater than that expressing ERbeta in 9 cases. Eight cases demonstrated only ERalpha mRNA hybridization signals in carcinoma cells. These results indicate that ERbeta is coexpressed with ERalpha in most ERbeta-positive breast carcinoma cells, which suggests that the expression of ERbeta depends on the presence of ERalpha in the great majority of human breast cancer. In addition, the number of carcinoma cases and/or the ratio of carcinoma cells expressing ERalpha was much greater than those expressing ERbeta. The relative ratio of ERalpha and ERbeta expression in carcinoma cells may be related to various estrogen-dependent biological features of human breast cancer.     

Estrogen receptors alpha and beta in the rodent mammary gland

An obligatory role for estrogen in growth, development, and functions of the mammary gland is well established, but the roles of the two estrogen receptors remain unclear. With the use of specific antibodies, it was found that both estrogen receptors, ERalpha and ERbeta, are expressed in the rat mammary gland but the presence and cellular distribution of the two receptors are distinct. In prepubertal rats, ERalpha was detected in 40% of the epithelial cell nuclei. This decreased to 30% at puberty and continued to decrease throughout pregnancy to a low of 5% at day 14. During lactation there was a large induction of ERalpha with up to 70% of the nuclei positive at day 21. Approximately 60-70% of epithelial cells expressed ERbeta at all stages of breast development. Cells coexpressing ERalpha and ERbeta were rare during pregnancy, a proliferative phase, but they represented up to 60% of the epithelial cells during lactation, a postproliferative phase. Western blot analysis and sucrose gradient centrifugation confirmed this pattern of expression. During pregnancy, the proliferating cell nuclear antigen was not expressed in ERalpha-positive cells but was observed in 3-7% of ERbeta-containing cells. Because more than 90% of ERbeta-bearing cells do not proliferate, and 55-70% of the dividing cells have neither ERalpha nor ERbeta, it is clear that the presence of these receptors in epithelial cells is not a prerequisite for estrogen-mediated proliferation.     

Estrogen receptor beta in breast cancer

Estrogen is essential for normal growth and differentiation in the mammary gland. It also supports growth of approximately 50% of primary breast cancers. For this reason, removal of estrogen or blocking of its action with the anti-estrogen, tamoxifen, is the main treatment for estrogen receptor alpha (ERalpha)-positive tumors. In 1996, when oncologists became aware of a second ER, ERbeta, there was some doubt as to whether this receptor would be of importance in breast cancer because the clinical consensus was that responsiveness to tamoxifen is related to the presence of ERalpha in breast cancer. Today we know that ERalpha and ERbeta have distinct cellular distributions, regulate separate sets of genes and can oppose each other's actions on some genes. We also know that ERbeta is widely expressed in both the normal and malignant breast and that there are proliferating cells in the breast which express ERbeta. In this review we summarize what is known about ERbeta in breast cancer and examine the possibility that ERbeta-selective ligands may well represent a useful class of pharmacological tools with a novel target, namely proliferating cells expressing ERbeta.     

Hormonal regulation of estrogen receptor alpha and beta gene expression in human granulosa-luteal cells in vitro

Estrogen is one of the major sex steroid hormones that is produced from the human ovary, and its actions are established to be a receptor-mediated process. Despite the demonstration of estrogen receptor (ER) expression, little is known regarding the regulation of ER in the human ovary. In the present study we investigated the expression and hormonal regulation of ERalpha and ERbeta in human granulosa-luteal cells (hGLCs). Using RT-PCR amplification, both ERalpha and ERbeta messenger ribonucleic acid (mRNA) were detected from hGLCs. Northern blot analysis revealed that ERalpha is expressed at a relatively lower level than ERbeta. Basal expression studies indicated that ERalpha mRNA levels remain unchanged, whereas ERbeta mRNA levels increased with time in culture in vitro, suggesting that ERbeta is likely to play a dynamic role in mediating estrogen action in hGLCs. The regulation of ERalpha and ERbeta expression by hCG was examined. hCG treatment (10 IU/mL) significantly attenuated the ERalpha (45%; P < 0.01) and ERbeta (40%; P < 0.01) mRNA levels. The hCG-induced decrease in ERalpha and ERbeta expression was mimicked by 8-bromo-cAMP (1 mmol/L) and forskolin (10 micromol/L) treatment. Additional studies using a specific protein kinase A (PKA) inhibitor (adenosine 3',5'-cyclic monophosphorothioate, Rp-isomer, triethylammonium salt) and an adenylate cyclase inhibitor (SQ 22536) further implicated the involvement of the cAMP/PKA signaling pathway in hCG action in these cells. The hCG-induced decrease in ERalpha and ERbeta mRNA levels was prevented in the presence of these inhibitors. Next, the effect of GnRH on ER expression was studied. Sixty-eight percent (P < 0.001) and 60% (P < 0.001) decreases in ERalpha and ERbeta mRNA levels, respectively, were observed after treatment with 0.1 micromol/L GnRH agonist (GnRHa). Pretreatment of the cells with a protein kinase C (PKC) inhibitor (GF109203X) completely reversed the GnRHa-induced down-regulation of ERalpha and ERbeta expression, suggesting the involvement of PKC in GnRH signal transduction in hGLCs. In agreement with the semiquantitative RT-PCR results, Western blot analysis detected a decrease in ERalpha and ERbeta proteins levels in hGLCs after treatment with hCG (10 IU/mL), GnRH (0.1 micromol/L), 8-bromo-cAMP (1 mmol/L), forskolin (10 micromol/L), or phorbol 12-myristate 13 acetate (10 micromol/L). Functionally, we demonstrated an inhibition of progesterone production in hGLCs in vitro by 17beta-estradiol, and this inhibitory effect was eliminated by pretreatment of 10 IU/mL hCG or 0.1 micromol/L GnRHa for 24 h before 17beta-estradiol administration. In summary, we observed a differential expression of ERalpha and ERbeta mRNA in hGLCs in vitro. The demonstration of hCG- and GnRHa-induced down-regulation of ERalpha and ERbeta gene expression suggests that hCG and GnRH may contribute to the control of granulosa-luteal cell function. Furthermore, our data suggest that the effects of hCG and GnRH on ERalpha and ERbeta expression in hGLCs are mediated in part by activation of PKA and PKC signaling pathways, respectively.     

Critical role for estrogen receptor alpha in negative feedback regulation of gonadotropin-releasing hormone mRNA expression in the female mouse

Estrogen exerts an important regulatory influence upon the functioning of the gonadotropin-releasing hormone (GnRH) neurons. Whether this is mediated by estrogen receptor alpha (ERalpha) or ERbeta or both ERs is presently unclear. Using female mice with targeted disruptions of ERalpha and ERbeta (alphaERKO and betaERKO, respectively) we have investigated the in vivo role of the two ERs in the negative feedback influence of estrogen upon GnRH mRNA expression. Compared with intact wild-type mice, plasma luteinizing hormone (LH) levels were substantially (p < 0.01) higher in intact alphaERKO females and increased modestly (p < 0.05) in intact betaERKO mice. Three weeks after ovariectomy, LH concentrations were elevated significantly in wild-type (p < 0.01) and betaERKO (p < 0.05) mice but not changed in alphaERKO females. Quantitative analysis of GnRH mRNA expression using in situ hybridization revealed that cellular GnRH mRNA content was greater (p < 0.05) in intact alphaERKO mice compared with intact wild-type and betaERKO mice. Following ovariectomy, GnRH mRNA expression was elevated in wild-type (p = 0.06) and betaERKO (p < 0.05) females but not alphaERKO mice. These data demonstrate that both ERalpha and ERbeta are involved in inhibiting LH levels at times of estrogen-negative feedback in vivo. However, only ERalpha appears to be critical for the estrogen-negative feedback suppression of GnRH mRNA expression in the female mouse.     

Expression of estrogen receptor subtypes in rat pituitary gland during pregnancy and lactation

The aim of this study was to examine whether the expression levels of mRNA of the three estrogen receptor (ER) subtypes, ERalpha, ERbeta, and truncated ER product-1 (TERP-1) found in the rat pituitary gland were modified during gestation, lactation, and postlactation periods. By using relative quantitative RT-PCR, we found that ERalpha mRNA significantly peaked in midpregnancy. However, the ERalpha protein level remained constant. ERbeta gene expression did not change throughout pregnancy, suggesting that it was not related to estradiol levels during this reproductive period. In contrast, both TERP-1 mRNA and protein levels dramatically increased throughout the second half of gestation, being faintly detectable in early pregnancy. TERP-1 expression was rapidly reversed by lactation, whereas neither pituitary ERalpha nor ERbeta relative levels were significantly altered. In addition, pup removal for 24-96 h on d 9 postpartum significantly reduced the expression of both ERalpha and ERbeta mRNA compared with that in lactating animals, but the expression of TERP-1 mRNA was no longer detected. Collectively, our data indicate that 1) TERP-1, ERalpha, and ERbeta expression levels are differentially regulated in the pituitary; 2) TERP-1 is variably expressed depending on the hormonal environment related to the estrous cycle, pregnancy, and lactation; and 3) TERP-1/ERalpha ratios dramatically change depending on reproductive periods, suggesting a critical role for TERP-1 in reproductive events.     

Estrogen receptors and proliferation markers in primary and recurrent breast cancer

To elucidate the clinical importance of estrogen receptor (ER) beta in breast cancer, 29 archival primary breast cancer specimens, six locally recurrent cancers, and five benign mammary tumors were examined histochemically for ERalpha, ERbeta and the proliferation markers Ki67 and cyclin A. In benign tumors, most epithelial cells contained ERbeta, but ERalpha was rare. In primary cancers, both ERalpha and ERbeta occurred in epithelial cells, the presence of ERbeta being associated with elevated expression of Ki67 and cyclin A, and ERalpha with decreased levels. Thus, the highest content of proliferation markers was seen in primary cancers that were ERalpha(-) ERbeta(+). Most Ki67-containing cells coexpressed ERbeta, but few showed ERalpha. In locally recurring cancers, ERalpha, ERbeta, and Ki67 were more highly expressed than in the corresponding primary tumors, and many cells containing ERbeta, but few with ERalpha, expressed Ki67. Surprisingly, ERbeta, but not ERalpha, was seen in the stromal cells of both primary and recurrent cancers. Because the response of breast cancers to tamoxifen therapy is correlated with the presence of ERalpha, cancer cells that lack ERalpha but contain ERbeta and proliferation markers represent a novel population of apparently proliferating cells that probably are not targeted by the current antiestrogens. Thus, appropriate ERbeta-specific ligands, perhaps in combination with tamoxifen, may be useful in improving the treatment of breast cancers.     

ERbeta1 and the ERbeta2 splice variant (ERbetacx/beta2) are expressed in distinct cell populations in the adult human testis

Estrogens can regulate germ cell function. Estrogen action is mediated via high affinity ERs; two subtypes (ERalpha and ERbeta) have been identified. We have shown previously that ERbeta is expressed in nuclei of multiple human testicular cells. A variant isoform of human (h) ERbeta (hERbetacx/2), formed by alternative splicing, has been identified in testicular cDNA libraries by two laboratories. The present study examined the expression of wild-type (ERbeta1) and variant (ERbeta2) beta receptors in human testes by 1) RT-PCR with isoform specific primers, and 2) single and double immunohistochemistry using monoclonal antibodies raised against peptides unique to the C termini of hERbeta1 and hERbeta2. PCR products specific for ERbeta1 and ERbeta2 were amplified from cDNA pools prepared from human testes and granulosa cells. On Western blots, the anti-ERbeta1 monoclonal antibody bound to recombinant ERbeta1 and the anti-ERbeta2 monoclonal to recombinant hERbeta2. Neither bound to the other ERbeta isoform nor to recombinant ERalpha. ERbeta1 and ERbeta2 proteins were both detected in human testis. Immunoexpression of ERbeta1 was most intense in pachytene spermatocytes and round spermatids, whereas low levels of expression were detected in Sertoli cells, spermatogonia, preleptotene, leptotene, zygotene, and diplotene spermatocytes. Highest levels of expression of ERbeta2 protein were detected in Sertoli cells and spermatogonia with low/variable expression in preleptotene, pachytene, and diplotene spermatocytes. No immunostaining was detected in elongating spermatids. Most interstitial cells expressed more ERbeta2 than ERbeta1. It is speculated that the cells most susceptible to modulation by estrogenic ligands are round spermatids in which levels of expression of ERbeta1 are high. In contrast, expression of ERbeta2, an isoform that may act as a dominant negative inhibitor of ER action, in Sertoli cells and spermatogonia, could protect these cells from adverse effects of estrogens.     

Expression and role of estrogen receptor alpha and beta in medullary thyroid carcinoma: different roles in cancer growth and apoptosis

Medullary thyroid carcinoma (MTC) originates from parafollicular C cells. Estrogen receptor beta(ERbeta) expressionwas detected in normal parafollicular C cells and MTC tumor tissue, but ERalpha expression in MTC tumors still remains undetermined. The appearance and loss of ERalpha or ERbeta expression has been known to play a role in the development and progression of many human cancers. We performed immunohistochemical studies of ERalpha, ERbeta, and Ki67, a mitotic index, in 11 human MTC tissue samples. ERalpha was detected in 10 cases (91%), and ERbeta expression was observed in 8 cases (72.7%). A majority (8/10) of ERalpha-positive tumors showing ERbeta Ki67 expression was detected in three cases (27.3%). Neither clinical parameters nor tumor node metastasis (TNM) tumor staging was correlated with the positivity for ERs or Ki67. To investigate the biological role of each ER, we used ER-negative MTC TT cells and adenoviral vectors carrying ERalpha (Ad-ERalpha), ERbeta (Ad-ERbeta), estrogen response element (ERE)-Luc (Ad-ERE-Luc), and activator protein 1 (AP1)-Luc (Ad-AP1-Luc). Estrogen stimulated and anti-estrogen, ICI 182 780, suppressed ERE reporter activity in TT cells expressing ERalpha or ERbeta, suggesting that both ERs use the same classical ERE-mediated pathway. Ad-ERalpha infection stimulated TT cell growth; in contrast, Ad-ERbeta infection suppressed their growth. Apoptosis was detected in Ad-ERbeta-infected TT cells. Estrogen and anti-estrogen suppressed AP1 activity in Ad-ERalpha-infected cells, whereas upon Ad-ERbeta infection estrogen further stimulated AP1 activity which in turn is suppressed by anti-estrogen, suggesting that each ER acts differently through a non-ERE-mediated pathway. Our results suggest that ERalpha and ERbeta may play different roles in MTC tumor growth and progression.     

Wild-type estrogen receptor (ERbeta1) and the splice variant (ERbetacx/beta2) are both expressed within the human endometrium throughout the normal menstrual cycle

Estrogen action is mediated via two subtypes of the estrogen receptor (ER), usually referred to as ERalpha and ERbeta. We have previously compared the spatial and temporal expressions of ERalpha and ERbeta proteins in human endometrium and reported that endothelial cells exclusively express ERbeta. In the present study we have extended our investigations to compare the pattern of expression of wild-type (ERbeta1) and a newly identified ERbeta variant isoform (ERbetacx/beta2) that lacks the ability to bind steroids. mRNAs encoding both ERbeta1 and ERbetacx/beta2 receptors were identified in human endometrial extracts by RT-PCR. Quantitative TaqMan R-TPCR demonstrated that levels of total mRNAs were increased significantly premenstrually as circulating progesterone levels declined. ERbeta1 and ERbetacx/beta2 proteins were identified within multiple cell types within the endometrium using isotype-specific monoclonal antibodies; immunoexpression of ERbetacx/beta2 appeared less intense than that of ERbeta1 in endometrial glandular epithelium and endothelial cells. Immunoexpression of ERbeta1 appeared unchanged throughout the menstrual cycle. In contrast, levels of ERbetacx/beta2-specific immunoreactivity were specifically reduced in gland cells within the functional layer, but not in those of the basal layer, in the midsecretory phase. It is possible that coexpression of ERbetacx/beta2 in cells containing ERbeta1 and/or ERalpha may modulate the effects of estrogens on the endometrium.     

17Beta-estradiol up-regulates vascular endothelial growth factor receptor-2 expression in human myometrial microvascular endothelial cells: role of estrogen receptor-alpha and -beta

Estrogen has a cardiovascular protective role in women due in part to its effect on the vasculature. The roles of the two estrogen receptors (ERs), ERalpha and ERbeta, in the vascular actions of estrogen are unclear, as are effects of estrogen on microvascular endothelial cells (MEC) derived from sex steroid-responsive tissues. The present study demonstrates that 17beta-estradiol, but not progesterone, increases vascular endothelial growth factor (VEGF) receptor (VEGFR) expression on human myometrial MEC measured using biotin-recombinant human (rh) VEGF(165) and flow cytometry. This response occurred in a time- and dose-dependent manner, with significantly increased rhVEGF(165) binding at 3 h and maximal responses between 0.1 and 10 nmol/liter 17beta-estradiol, which was blocked by the antiestrogen ICI 182,780. Approximately 60% of samples demonstrated this response to 17beta-estradiol. All samples of myometrial MEC expressed both ERbeta mRNA and protein demonstrated by semiquantitative RT-PCR and Western blotting. However, ERalpha mRNA and protein were expressed in only 13 of 21 MEC samples. There was a significant association between ERalpha expression in myometrial MEC and their ability to respond to 17beta-estradiol by increasing rhVEGF(165) binding. 17beta-estradiol increased VEGFR-2 expression in ERalpha-expressing MEC isolates, which also demonstrated increased rhVEGF(165) binding, but failed to have these effects on ERalpha negative samples. Similarly, 17beta-estradiol augmented VEGF-induced MEC proliferation in ERalpha-expressing MEC samples, which was blocked by ICI 182,780. These observations suggest that 17beta-estradiol increases VEGFR-2 expression on human myometrial MEC promoting endothelial cell proliferation, an effect that varies between subjects and appears to be mediated primarily by ERalpha.