Estrogen receptor-alpha (ERalpha), but not ERbeta,modulates estrogen stimulation of the ERalpha-truncated variant,TERP-1

Estrogens regulate pituitary gene expression through two nuclear receptors (ERs), ERalpha and ERbeta. Rodent pituitary also expresses high levels of the pituitary-specific ERalpha isoform, truncated ER product-1 (TERP-1), which modulates the response of both ER forms to 17beta-estradiol (E2). Under physiological conditions, E2 stimulates TERP-1 expression from an ERalpha intronic promoter containing several potential binding sites for ERs. To evaluate the role of intact ER proteins on TERP-1 expression, we measured basal expression and steroid stimulation of TERP-1 in wild-type (WT) mice and mice in which either the ERalpha (ERalphaKO) or the ERbeta (ERbetaKO) gene was disrupted. TERP-1 mRNA expression was assessed by semiquantitative RT-PCR, and protein expression was evaluated by immunoblots. Both TERP-1 mRNA and protein were expressed in pituitaries from castrate WT, ERalphaKO, and ERbetaKO male and female mice. E2 stimulated TERP-1 mRNA expression in WT and ERbetaKO mice of both sexes, but had no effect on TERP-1 mRNA in either male or female ERalphaKO mice. Testosterone treatment also stimulated TERP-1 in WT, ERalphaKO, and ERbetaKO male mice. We conclude that ERalpha is critical for E2 stimulation, but not basal expression, of the TERP promoter, and that testosterone may act through the androgen receptor to stimulate the TERP-1 promoter in males.     

Both estrogen and raloxifene cause G1 arrest of vascular smooth muscle cells

The proliferation of vascular smooth muscle cells (VSMC) is a crucial pathophysiological process in the development of atherosclerosis. Although estrogen is known to inhibit the proliferation of VSMC, the mechanism responsible for this effect remains to be elucidated. In addition, the effect of raloxifene on VSMC remains unknown. We have shown here that 17beta-estradiol (E(2)) and raloxifene significantly inhibited the platelet-derived growth factor (PDGF)-stimulated proliferation of cultured human VSMC. Flow cytometry demonstrated that PDGF-stimulated S-phase progression of the cell cycle in VSMC was also suppressed by E(2) or raloxifene. We found that PDGF-induced phosphorylation of retinoblastoma protein (pRb), whose hyperphosphorylation is a hallmark of the G1-S transition in the cell cycle, was significantly inhibited by E(2) and raloxifene. These effects were associated with a decrease in cyclin D1 expression, without a change in cyclin-dependent kinase 4 or cyclin-dependent kinase inhibitor, p27(kip1) expression. ICI 182,780 abolished the inhibitory effects of E(2) and raloxifene on PDGF-induced pRb phosphorylation. Next, we examined which estrogen receptor (ER) is necessary for these effects of E(2) and raloxifene. Since VSMC express both ERalpha and ERbeta, A10, a rat aortic smooth muscle cell line that expresses ERbeta but not ERalpha, was used. The dose-dependent stimulation of A10 cell proliferation by PDGF was not inhibited by E(2) or raloxifene in contrast to the results obtained in VSMC. Moreover, E(2) and raloxifene significantly inhibited the PDGF-induced cyclin D1 promoter activity in A10 cells transfected with cDNA for ERalpha but not in the parental cells. These results suggested that E(2) and raloxifene exert an antiproliferative effect in VSMC treated with PDGF, at least in part through inhibition of pRb phosphorylation, and that the inhibitory effects of E(2) and raloxifene may be mainly mediated by ERalpha.     

Inducible upregulation of oestrogen receptor-beta1 affects oestrogen and tamoxifen responsiveness in MCF7 human breast cancer cells

To investigate the effect of altered oestrogen receptor (ER)alpha and ERbeta expression on oestrogen and anti-oestrogen action in breast cancer, we have stably expressed an inducible ERbeta1 in MCF7 breast cancer cells. Stably expressing clones were isolated and over-expression of ERbeta1 correlated with increased levels of specific radiolabelled oestradiol (E2) binding. Increased ERbeta1 did not affect endogenous levels of ERalpha but increased progesterone receptor (PR) levels. Over-expression of ERbeta1 reduced growth responses to E2 in contrast to little if any effect of over-expression of ERalpha. In oestrogen-replete conditions, over-expression of ERbeta1 but not ERalpha reduced proliferation. Over-expression of ERbeta1 did not result in anti-oestrogen resistance but was associated with increased sensitivity to 4-hydroxytamoxifen. Our results suggested that over-expression of ERbeta1 in the presence of an endogenously expressed ERalpha was associated with tamoxifen sensitivity but may negatively modulate ERalpha-mediated growth. However, not all ERalpha activities were inhibited since endogenous PR expression was increased by both ERalpha and ERbeta1 over-expression. These data paralleled those seen in some in vivo studies showing a relationship between PR and ERbeta expression as well as ERbeta expression and tamoxifen sensitivity of ER-positive breast cancer patients. These models are relevant and will be useful for dissecting the role of ERbeta1 expression in ER-positive breast cancer.     

Estrogen and raloxifene induce apoptosis by activating p38 mitogen-activated protein kinase cascade in synthetic vascular smooth muscle cells

Proliferation of vascular smooth muscle cells (VSMC) plays a major role as an initiating event of atherosclerosis. Although estrogen directly inhibits the proliferation of VSMC, the mechanism has not been firmly established. In addition, the effect of raloxifene on VSMC remains unknown. 17Beta-estradiol (E(2)) and raloxifene significantly inhibited the growth of VSMC under growth-stimulated conditions. Since mitogen-activated protein (MAP) kinases have been implicated in VSMC proliferation, the role of MAP kinases in both the E(2)- and raloxifene-induced growth inhibition of VSMC was studied. Both E(2) and raloxifene caused rapid, transient phosphorylation and activation of p38 that was not affected by actinomycin D and was blocked by ICI 182,780. In contrast with p38 phosphorylation, extracellular signal-regulated protein kinase (ERK) phosphorylation was significantly inhibited and c-Jun N-terminal kinase (JNK) phosphorylation was not changed by E(2). Because VSMC expressed both estrogen receptor (ER) alpha and ERbeta, it is not known which of them mediates the E(2)-induced phosphorylation of p38. Although E(2) did not affect the p38 phosphorylation in A10 smooth muscle cells, which express ERbeta but not ERalpha, transfection of ERalpha expression vector into A10 cells rendered them susceptible to induction of p38 phosphorylation by E(2). We then examined whether E(2) and raloxifene induce apoptosis through a p38 cascade. Both E(2) and raloxifene induced apoptosis under growth-stimulated conditions. The p38 inhibitor SB 203580 completely blocked the E(2)-induced apoptosis. Our findings suggest that both E(2)- and raloxifene-induced inhibition of VSMC growth is due to induction of apoptosis through a p38 cascade whose activation is mediated by ERalpha via a nongenomic mechanism.     

Genistein enhances insulin-like growth factor signaling pathway in human breast cancer (MCF-7) cells

Physiological concentration of genistein, a natural isoflavonoid phytoestrogen, stimulates human breast cancer (MCF-7) cells proliferation. In this study, we hypothesize that low concentration of genistein mimics the action of 17beta-estradiol in stimulation of MCF-7 cell growth by enhancement of IGF-I signaling pathway. Genistein, at 1 microM, stimulated the growth of MCF-7 cells. Cell cycle analysis showed that 1 micro M genistein significantly increased the S phase and decreased the G0G1 phase of MCF-7 cells. The protein and mRNA expression of IGF-I receptor (IGF-IR) and insulin receptor substrate (IRS)-1, but not Src homology/collagen protein, increased in response to 1 microM genistein in a time-dependent manner. These effects could be completely abolished by cotreatment of MCF-7 cells with estrogen antagonist ICI 182780 (1 microM) and tamoxifen (0.1 microM). Our results also showed that genistein induction of IGF-IR and IRS-1 expression resulted in enhanced tyrosine phosphorylation of IGF-IR and IRS-1 on IGF-I stimulation. Taken together, these data provide the first evidence that the IGF-IR pathway is involved in the proliferative effect of low-dose genistein in MCF-7 cells.     

Transcriptional profiling of estrogen-regulated gene expression via estrogen receptor (ER) alpha or ERbeta in human osteosarcoma cells: distinct and common target genes for these receptors

Estrogens exert many important effects in bone, a tissue that contains both estrogen receptors alpha and beta (ERalpha and ERbeta). To compare the actions of these receptors, we generated U2OS human osteosarcoma cells stably expressing ERalpha or ERbeta, at levels comparable with those in osteoblasts, and we characterized their response to 17beta-estradiol (E2) over time using Affymetrix GeneChip microarrays to determine the expression of approximately 12,000 genes, followed by quantitative PCR verification of the regulation of selected genes. Of the approximately 100 regulated genes we identified, some were stimulated by E2 equally through ERalpha and ERbeta, whereas others were selectively stimulated via ERalpha or ERbeta. The E2-regulated genes showed three distinct temporal patterns of expression over the 48-h time course studied. Of the functional categories of the E2-regulated genes, most numerous were those encoding cytokines and factors associated with immune response, signal transduction, and cell migration and cytoskeleton regulation, indicating that E2 can exert effects on multiple pathways in these osteoblast-like cell lines. Of note, E2 up-regulated several genes associated with cell motility selectively via ERbeta, in keeping with the selective E2 enhancement of the motility of ERbeta-containing cells. On genes regulated equally by E2 via ERalpha or ERbeta, the phytoestrogen genistein preferentially stimulated gene expression via ERbeta. These studies indicate both common as well as distinct target genes for these two ERs, and identify many novel genes not previously known to be under estrogen regulation.     

Estradiol regulates corticotropin-releasing hormone gene (crh) expression in a rapid and phasic manner that parallels estrogen receptor-alpha and -beta recruitment to a 3',5'-cyclic adenosine 5'-monophosphate regulatory region of the proximal crh promoter

In the central nervous system, CRH regulates several affective states. Dysregulation of neuronal crh expression in the paraventricular nucleus of the hypothalamus correlates with some forms of depression, and amygdalar crh expression may modulate levels of anxiety. Because estrogens modulate these states, we sought to determine 17beta-estradiol (E2) effects on crh expression. CRH mRNA levels were measured in the AR-5 amygdaloid cell line by RT-PCR analysis. They increased by 1 min of E2 treatment, suggesting that crh behaves as an immediate-early gene. After peaking at 3 min, CRH mRNA returned to basal levels and then increased by 60 min. To dissect some of the molecular mechanisms underlying these events, we measured occupancy of the crh promoter by estrogen receptors (ERs) and coactivators, using chromatin immunoprecipitation. Because this promoter does not contain palindromic estrogen response elements, we targeted the region of a cAMP regulatory element (CRE), implicated in crh regulation. The temporal pattern of the mRNA response was mimicked by recruitment of ERalpha and -beta, phospho-CRE-binding protein, coactivators steroid receptor coactivator-1 and CRE-binding protein-binding protein (CBP), and an increase in histone 3 and 4 acetylation. Lastly, ERalpha and -beta loading were temporally dissociated, peaking at 1 and 3 min, respectively. The ER peaks were associated with coactivators and acetylation patterns. ERalpha associated with phospho-CRE-binding protein, CBP, steroid receptor coactivator-1, and increased acetylated histone 3. ERbeta associated with CBP and increased acetylated histone 4. The tight temporal correlation between E2-induced CRH mRNA levels and promoter occupancy by ERs strongly suggest that E2 regulates crh expression through an ERalpha- and/or ERbeta-CRE alternate pathway.     

Differential regulation of estrogen receptor (ER)alpha and ERbeta in primate mammary gland

Estrogen, mainly estradiol (E2), and progesterone (P) are essential for the growth and differentiation of the breast, but their roles in breast cancer are highly debated. To understand how E2 and P influence cell proliferation and differentiation, it is essential to know how their receptors are regulated. Because of limited tissue availability, little is known about regulation of the two estrogen receptors (ERalpha and ERbeta) and the two progesterone receptor isoforms (PR-A and PR-B) in the normal human breast. What we know comes from rodent studies, which are not always pertinent for the human breast. We report now on regulation of gonadal hormone receptors during the menstrual cycle, pregnancy, and lactation in rhesus monkey mammary gland and on the relationship of these receptors to proliferation. We found that ERalpha but not ERbeta is down-regulated when E2 levels increase and when cells enter the cell cycle. PR-B but not PR-A is expressed in proliferating cells. Thus under normal conditions, the ratio of ERalpha to ERbeta in the breast depends on plasma concentrations of E2. Elevated expression of ERalpha (as occurs in postmenopausal women) is a normal response to loss of E2 and indicates nonproliferating cells. As selective receptor ligands become available, they will be helpful in delineation of the functions of these receptors.     

Identification of wild-type and exon 5 deletion variants of estrogen receptor beta in normal human mammary gland

We have examined messenger RNA (mRNA) expression of estrogen receptor (ER) alpha, wild-type ERbeta (mRNA and protein), and ERbeta exon 5 deletion variants (ERbeta delta5) in samples of normal human mammary gland obtained from 37 premenopausal subjects undergoing reduction mammoplasty. Comparing individual expression, ERbetaP mRNA predominated, expressed in 34 of 37 samples (91%), whereas ERalpha was found in 21 of 37 cases (57%). Receptor combinations were then analyzed and compared. Most samples either coexpressed ERalpha with ERbeta (54%) or expressed just ERbeta (38%). Immunohistochemical analysis revealed that ERbeta mRNA expression mirrored that of protein. Immunoreactivity was observed in the nucleus with additional evidence of cytoplasmic staining in those epithelial cells lining the breast ducts. Sporadic immunoreactivity was also detected in stromal cells. Expression of wild type and ERbeta delta5 was analyzed, and their association with ERalpha was compared. Most samples coexpressed wild-type ERbeta and the splice variant (62%; P = 0.05), with 30% exclusively expressing wild-type ERbeta. Although samples coexpressing wild type and variant ERbeta showed no statistical association with ERalpha, those samples expressing only wild-type ERP, showed a trend toward associations with ERalpha (P = 0.07). In conclusion, our data would support a role for ERbeta in the normal human mammary gland, where we propose it may be the dominant receptor.     

Decreased expression of e6-associated protein in breast and prostate carcinomas

The E6-associated protein (E6-AP) is a dual function protein. It acts as an E3 ubiquitin-protein ligase as well as a steroid hormone receptor coactivator. Considering the influence of steroid hormone receptors and their coactivators in the normal development and tumorigenesis of reproductive organs of both genders, we examined the roles of E6-AP in the tumorigenesis of breast and prostate tissues. We demonstrated that the expression of E6-AP protein is decreased in human invasive breast and prostate carcinomas compared with their adjacent normal tissues, and this down-regulation of E6-AP is accompanied by the up-regulation of estrogen receptor (ER)-alpha in breast and androgen receptor (AR) in prostate carcinomas. Furthermore, our in vivo data from E6-AP-knockout animals indicated that the expression levels of ERalpha and AR are increased in E6-AP-null mammary and prostate glands, respectively, when compared with that of normal control animals, suggesting that E6-AP modulates the protein levels of ERalpha in breast and AR in prostate glands.     

Low phosphorylation of estrogen receptor alpha (ERalpha) serine 118 and high phosphorylation of ERalpha serine 167 improve survival in ER-positive breast cancer

Endocrine therapy has become the most important treatment option for women with estrogen receptor (ER)-positive breast cancer. Urgently needed are prognostic assays that can identify those who need additional adjuvant therapy, such as signal transduction inhibitors or chemotherapy, for ER-positive early breast cancer. We examined phosphorylation of ERalpha serine (Ser) 118, ERalpha Ser167, p44/42 mitogen-activated protein kinase (MAPK), and Akt and expression of progesterone receptor, amplified in breast cancer 1 (AIB1), human epidermal growth factor receptor 2 (HER2), p53, and Ki67 in ER-positive breast cancers by immunohistochemistry, and analyzed their significance for prognosis. Phosphorylation levels of ERalpha Ser118, ERalpha Ser167, MAPK, and Akt were positively correlated. AIB1 expression was significantly associated with phosphorylation of ERalpha Ser118, MAPK, and Akt, and HER2 expression. Low phosphorylation of ERalpha Ser118 and high phosphorylation of ERalpha Ser167 were associated with significantly improved disease-free (P=0.0003 and P=0.0002 respectively) and overall survival (P=0.0007 and P=0.0016 respectively) in multivariate analyses. Our data suggest that phosphorylation of ERalpha Ser118 and ERalpha Ser167 affects survival in ER-positive breast cancer and could be helpful in distinguishing patients who are likely to benefit from endocrine therapy alone from those who are not.     

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.     

Stimulation of the novel estrogen receptor-alpha intronic TERP-1 promoter by estrogens, androgen, pituitary adenylate cyclase-activating peptide, and forskolin, and autoregulation by TERP-1 protein

The estrogen receptor-alpha (ERalpha) pituitary-specific variant, TERP-1, is regulated dramatically by physiological status. We examined hormonal regulation of the TERP-1 promoter in transient transfection assays in GH3 somatolactotrope cells. We found that 17beta-estradiol (E2), genistein, androgen, pituitary adenylate cyclase-activating peptide, and forskolin (FSK) all stimulated TERP-1 promoter activity, whereas progesterone had no effect. ERalpha bound to a palindromic estrogen response element (ERE) and two half-site EREs; mutation of any of these sites decreased basal expression and completely obliterated E2 stimulation. In contrast, mutation of an activator protein-1 site decreased basal and FSK-stimulated promoter activity, but not E2 or androgen stimulation. The pure antiestrogen ICI 182,780 suppressed E2 and genistein, but not FSK or androgen, stimulation. Similarly, mutation of the ERE palindrome or half-site EREs suppressed promoter stimulation by E2 and genistein, but not by androgen or FSK. Because TERP-1 levels regulate ERalpha function on model promoters, we tested TERP-1 modulation of its own and other physiological promoters. TERP-1 suppressed basal and E2-stimulated expression of its own promoter. TERP-1 suppression required the ERE regions of the promoter, and the dimerization domain of TERP-1. TERP-1 overexpression also suppressed E2 stimulation of the progesterone receptor and prolactin promoters. Thus, estrogens, androgen, and FSK can stimulate TERP-1 promoter activity, and increased TERP-1 expression modulates E2 stimulation of physiological promoters. These data suggest that TERP-1 regulation may play a significant role in modifying pituitary ERalpha responses.