ERbeta Subtype Research Articles

In vitro chronic administration of ERbeta selective ligands and prostate cancer cell growth: hypotheses on the selective role of 3beta-adiol in AR-positive RV1 cells.

Prostate cancer (PC) progression from androgen-dependent (AD) to castration-resistant (CR) disease is a process caused by modifications of different signal transduction pathways within tumor microenvironment. Reducing cell proliferation, estrogen receptor beta (ERbeta) is emerging as a potential target in PC chemoprevention. Among the known selective ERbeta ligands, 3beta-Adiol, the endogenous ligand in the prostate, has been proved to counteract PC progression. This study compares the effects of chronic exposure (1–12 weeks) to different ERbeta selective ligands (DPN, 8beta-VE2, 3beta-Adiol) on proliferation of human androgen-responsive CWR22Rv1 cells, representing an intermediate phenotype between the AD- and CR-PC. 3beta-Adiol (10 nM) is the sole ligand decreasing cell proliferation and increasing p21 levels. In vitro transcriptional activity assays were performed to elucidate different behavior between 3beta-Adiol and the other ligands; in these experiments the endogenous and the main ERbeta subtype activation were considered. It is concluded that ERbeta activation has positive effects also in androgen-responsive PC. The underlying mechanisms are still to be clarified and may include the interplay among different ERbeta subtypes and the specific PC microenvironment. ERbeta agonists might be useful in counteracting PC progression, although the final outcome may depend upon the molecular pattern specific to each PC lesion.

Activation of estrogen receptor β-dependent nitric oxide signaling mediates the hypotensive effects of estrogen in the rostral ventrolateral medulla of anesthetized rats

BackgroundApart from their well-known peripheral cardiovascular effects, emerging evidence indicates that estrogen acts as a modulator in the brain to regulate cardiovascular functions. The underlying mechanisms of estrogen in central cardiovascular regulation, however, are poorly understood. The present study investigated the cardiovascular effects of 17β-estradiol (E2β) in the rostral ventrolateral medulla (RVLM), where sympathetic premotor neurons are located, and delineated the engagement of nitric oxide (NO) in E2β-induced cardiovascular responses.MethodsIn male Sprague-Dawley rats maintained under propofol anesthesia, the changes of blood pressure, heart rate and sympathetic vasomotor tone after microinjection bilaterally into the RVLM of a synthetic estrogen, E2β were examined for at least 120 min. The involvement of ERα and/or ERβ subtypes was determined by microinjection of selective ERα or ERβ agonist into bilateral RVLM. Different NO synthase (NOS) inhibitors were used to evaluate the involvement of differential of NOS isoforms in the cardiovascular effects of E2β.ResultsBilateral microinjection of E2β (0.5, 1, or 5 pmol) into the RVLM dose-dependently decreased systemic arterial pressure (SAP) and the power density of the vasomotor components of SAP signals, our experimental index for sympathetic neurogenic vasomotor tone. These cardiovascular depressive effects of E2β (1 pmol) were abolished by co-injection of ER antagonist ICI 182780 (0.25 or 0.5 pmol), but not a transcription inhibitor actinomycin D (10 nmol). Like E2β, microinjection bilaterally into the RVLM of a selective ERβ agonist 2,3-bis(4-hydroxyphenyl) propionitrile (DPN, 1, 2, or 5 pmol) induced significant decreases in these hemodynamic parameters in a dose-dependent manner. In contrast, the selective ERα agonist 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole (5 pmol) did not influence the same cardiovascular parameters. Co-administration bilaterally into the RVLM of NOS inhibitor NG-nitro-L-arginine methyl ester (5 nmol) or selective inducible NOS (iNOS) inhibitor S-methylisothiourea (25 pmol), but not selective neuronal NOS inhibitor 7-nitroindazole (0.5 pmol) or endothelial NOS inhibitor N5-(1-Iminoethyl)-L-ornithine (2.5 pmol), significantly attenuated the cardiovascular depressive effects elicited by DPN (2 pmol).ConclusionOur results indicate that E2β in the RVLM elicited short-term cardiovascular depressive effects via an ERβ-dependent nontranscriptional mechanism. These vasodepressor effects of E2β are likely to be mediated by the iNOS-derived NO in the RVLM.

Raloxifene plus 17beta-estradiol inhibits proliferation of primary cultured vascular smooth muscle cells and human mammary endothelial cells via the janus kinase/signal transducer and activator of transcription3 cascade

Long-term use of estrogen replacement therapy increases the risk of breast cancer. Presently, we investigated the effects and mechanisms of Raloxifene, a second generation selective estrogen receptor modulator, plus 17beta-estradiol on the proliferation of primary cultured vascular smooth muscle cells (VSMC) and human mammary endothelial cells (HMEC). Raloxifene plus 17beta-estradiol inhibited angiotensinII-induced VSMC proliferation and rapid phosphorylation of STAT 3; these effects were blocked by AG490, the janus kinase/signal transducer and activator of transcription3 (JAK/STAT 3) inhibitor. STAT 3 production was not affected. In primary cultured HMEC, immunofluorescence identified the ERbeta subtype, but not the ERalpha subtype, in the nucleus. Raloxifene plus 17beta-estradiol inhibited 17beta-estradiol-induced proliferation of HMEC. Western blot analysis established that Raloxifene attenuated the 17beta-estradiol-induced phosphorylation of STAT 3, and that this effect was blocked by AG490. We conclude that Raloxifene plus 17beta-estradiol inhibits the proliferation of VSMC and HMEC through the JAK/STAT 3 cascade, which in primary cultured HMEC may be implemented through ERbeta.

Estrogen Receptor α Mediates Acute Potassium Channel Stimulation in Human Coronary Artery Smooth Muscle Cells

The pleiotropic effects of estrogen are mediated via stimulation of two estrogen receptor (ER) subtypes, ERalpha and ERbeta. Although a number of studies have identified expression of one or both subtypes in estrogen target tissues, fewer studies have correlated ER expression with a functional role of these proteins in regulating cellular excitability. In the present study, we have combined cellular fluorescence, immunocytochemistry, and molecular expression techniques with single-channel patch-clamp studies to determine which ER mediates estrogen-stimulated potassium channel activity in human coronary artery smooth muscle cells (HCASMC). We had demonstrated previously that estrogen stimulates activity of the large-conductance, calcium- and voltage-activated potassium (BK(Ca)) channel in HCASMC via a nongenomic mechanism. We now demonstrate expression of both ERalpha and ERbeta subtypes in HCASMC. Functionally, however, expression of ERalpha antisense plasmid abolished the acute effect of estrogen on these channels, whereas estrogen retained its ability to stimulate BK(Ca) channels in cells transfected with only green fluorescence protein. In contrast, overexpression of ERalpha enhanced the stimulatory action of estrogen in HCASMC. Transfection with ERalpha antisense/sense plasmid did not alter ERbeta expression. These findings indicate that the ERalpha isoform mediates estrogen-induced stimulation of BK(Ca) channels in HCASMC and thereby provide evidence for a receptor-dependent signaling mechanism that can mediate estrogen-induced inhibition of cellular excitability.

Differential Response of Estrogen Receptor Subtypes to 1,3-Diarylindene and 2,3-Diarylindene Ligands

Estrogen receptors (ERs) control transcription of genes important for normal human development and reproduction. The signaling networks are complex, and there is a need for a molecular level understanding of the roles of receptor subtypes ERalpha and ERbeta in normal physiology and as therapeutic targets. We synthesized two series of ER ligands, based on a common indene scaffold, in an attempt to develop compounds that can selectively modulate ER-mediated transcription. The 3-ethyl-1,2-diarylindenes, utilizing an amide linker for the 1-aryl extension, bind weakly to the ERs. The 2,3-diarylindenes bind with high affinity to the ER subtypes and demonstrate a range of different biological activities, both in transcriptional reporter gene assays and inhibition of estradiol-stimulated proliferation of MCF-7 cells. Several ligands differentiate between ERalpha and ERbeta subtypes at an estrogen response element (ERE), displaying various levels of partial to full agonist activity at ERalpha, while antagonizing estradiol action at ERbeta.

Indazole Estrogens: Highly Selective Ligands for the Estrogen Receptor β

The estrogen receptors, ERalpha and ERbeta, are important pharmaceutical targets. To develop ERbeta-selective ligands, we synthesized a series of nonsteroidal compounds having a phenyl-2H-indazole core with different groups at C-3. Several of these show high affinity and good ERbetaselectivity, especially those with polar and/or polarizable substituents at this site (halogen, CF(3), nitrile); the best compounds have affinities for ERbeta comparable to estradiol, with ERbetaaffinity selectivity >100. This potency and ERbeta selectivity is also seen in cell-based transcriptional assays, where several compounds showed ERbeta efficacies equivalent to that of estradiol with ERbeta potency selectivities of 100. These compounds might prove useful as selective pharmacological probes to study the biological actions of estrogens mediated through ERbeta, and they might lead to the development of useful pharmaceuticals. These findings also contribute to an evolving pharmacophore that characterizes certain nonsteroidal ligands having high ERbeta subtype affinity and potency selectivity.

Estrogen receptor-beta is the predominant estrogen receptor subtype in human oral epithelium and salivary glands.

Many studies have shown that the oral mucosa and salivary glands are sensitive to estrogen action. However, the expression of estrogen receptors (ERs) within these tissues is an area of controversy. ERs exist as two subtypes (ERalpha and ERbeta), and we hypothesized that the incongruity between ER expression and estrogen sensitivity may result from differential expression of ER subtypes in oral tissues. To test this hypothesis, we analyzed oral mucosal and salivary gland samples for ERalpha and ERbeta protein expression by immunohistochemistry from a cross-section of patients attending hospital for surgical problems of the head and neck. ERalpha was not detected in oral buccal and gingival epithelium or in salivary glands. In contrast, ERbeta was widely expressed at high levels in all oral tissues studied. Within these tissues, ERbeta was observed primarily in keratinocytes and salivary gland acinar and ductal cells. Our results demonstrating the expression of only the ERbeta subtype within oral tissues may explain the contradictory results from previous studies investigating ER expression in these tissues. Importantly, these results suggest that estrogens may act via ERbeta in oral tissues and explain the effect of hormonal changes on the oral mucosa as well as on saliva secretion and composition.

A new series of estrogen receptor modulators that display selectivity for estrogen receptor beta.

A series of 1,3,5-triazine-based estrogen receptor (ER) modulators that are modestly selective for the ERbeta subtype are reported. Compound 1, which displayed modest potency and selectivity for ERbeta vs ERalpha, was identified via high-throughput screening utilizing an ERbeta SPA-based binding assay. Subsequent analogue preparation resulted in the identification of compounds such as 21 and 43 that display 25- to 30-fold selectivity for ERbeta with potencies in the 10-30 nM range. These compounds profile as full antagonists at ERbeta and weak partial agonists at ERalpha in a cell-based reporter gene assay. In addition, the X-ray crystal structure of compound 15 complexed with the ligand binding domain of ERbeta has been solved and was utilized in the design of more conformationally restrained analogues such as 31 in an attempt to increase selectivity for the ERbeta subtype.

SERMs: how do they work?

characterisation of distinct ER subtypes. Most notable is the identification of the ER-alpha and ER-beta subtypes, but additional ER species, as well as receptor mechanisms via non-classical pathways, have been suggested by recent work. Dimerisation of the ER, an essential element of the ‘classical’ ER action pathway, has recently been shown to occur not only with ERalpha and ER-beta homodimers, but also with heterodimers involving both receptor subtypes. The distribution through various body tissues of ER subtypes adds an additional layer of complexity to the situation. A level of complexity that is just beginning to emerge involves ER regulatory proteins. Classical ER action involves a number of cellular regulatory proteins, many of which bind to ER complexes. Alterations of ER conformation lead to alterations of such interactions. This pathway clearly contributes to alterations in ER mechanistic pathways, in ways that have thus far been characterised in only a few instances.

Estrogen-Related Abnormalities in Glomerulosclerosis-Prone Mice: Reduced Mesangial Cell Estrogen Receptor Expression and Prosclerotic Response to Estrogens

The development and progression of glomerulosclerosis (GS) is determined by the genetic background. The incidence of end-stage renal disease is increased in postmenopausal women, suggesting that estrogen deficiency may play a role in the accumulation of extracellular matrix by mesangial cells (MCs), which are primarily responsible for the synthesis and degradation of this matrix. Using mouse models that are prone or resistant to the development of GS, we compared the expression of estrogen receptor (ER)-alpha and ER-beta subtypes in GS-prone and GS-resistant glomeruli and isolated MCs, and examined the effects of estrogens on ER, collagen, and matrix metalloproteinase (MMP) expression in MCs. Glomeruli and MCs from GS-prone mice had decreased expression of ER-alpha and ER-beta subtypes and ER transcriptional activity was also decreased in their MCs. Importantly, although 17 beta-estradiol treatment resulted in decreased collagen accumulation and increased MMP-9 expression and activity in MCs from GS-resistant mice, there was, paradoxically, no effect on collagen accumulation and decreased MMP-9 expression and activity in MCs from GS-prone mice. Thus, GS susceptibility is associated with diminished ER expression in MCs. The renal protective effects of estrogens, including decreased collagen accumulation and increased MMP-9 expression, seem to be blunted in GS-prone MCs.

Pyrazole ligands: structure-affinity/activity relationships and estrogen receptor-alpha-selective agonists.

We have found that certain tetrasubstituted pyrazoles are high-affinity ligands for the estrogen receptor (ER) (Fink et al. Chem. Biol. 1999, 6, 205-219) and that one pyrazole is considerably more potent as an agonist on the ERalpha than on the ERbeta subtype (Sun et al. Endocrinology 1999, 140, 800-804). To investigate what substituent pattern provides optimal ER binding affinity and the greatest enhancement of potency as an ERalpha-selective agonist, we prepared a number of tetrasubstituted pyrazole analogues with defined variations at certain substituent positions. Analysis of their binding affinity pattern shows that a C(4)-propyl substituent is optimal and that a p-hydroxyl group on the N(1)-phenyl group also enhances affinity and selectivity for ERalpha. The best compound in this series, a propylpyrazole triol (PPT, compound 4g), binds to ERalpha with high affinity (ca. 50% that of estradiol), and it has a 410-fold binding affinity preference for ERalpha. It also activates gene transcription only through ERalpha. Thus, this compound represents the first ERalpha-specific agonist. We investigated the molecular basis for the exceptional ERalpha binding affinity and potency selectivity of pyrazole 4g by a further study of structure-affinity relationships in this series and by molecular modeling. These investigations suggest that the pyrazole triols prefer to bind to ERalpha with their C(3)-phenol in the estradiol A-ring binding pocket and that binding selectivity results from differences in the interaction of the pyrazole core and C(4)-propyl group with portions of the receptor where ERalpha has a smaller residue than ERbeta. These ER subtype-specific interactions and the ER subtype-selective ligands that can be derived from them should prove useful in defining those biological activities in estrogen target cells that can be selectively activated through ERalpha.

Steroids and the endometrium.

Steroids are commonly employed in current clinical practice. The benefits of steroids in hormone replacement therapy, contraception and prevention or treatment of breast cancer are limited by their side effects arising from disorders in endometrial function. These side effects are complex and enclose bleeding problems and endometrial proliferation during hormone replacement therapy and antioestrogen treatment or menstrual disturbances during oral contraception. Numerous reports have identified gene targets influenced by steroids and have implicated these products as contributors to endometrial physiology or pathology. The expression of estrogen and progesterone receptors is regulated by steroids. The new estrogen receptor (ER) subtype ERbeta with different functional characteristics from ERalpha was recently described in endometrium. In addition, there is now increasing evidence that the functionally distinct progesterone receptor (PR) isoforms A and B are differentially expressed in this tissue. The relative proportions of these steroid receptors and their interaction determine the expression of specific genes upon steroidal stimulation. Steroids induce endometrial expression of various growth and angiogenic factors. Dysregulations of this steroid modulated expression is believed to be involved in the pathogenesis of many endometrial diseases. Irregular bleeding induced by steroidal contraception, for example, is thought to involve aberrant endometrial vascular development and expression of angiogenic growth factors. The antioestrogen tamoxifen induces growth factors like vascular endothelial growth factor and adrenomedullin which may be key mediators of endometrial neoplastic effects. This review describes recent advances regarding the mechanism of action of steroids on endometrium. The expression of oestrogen and progesterone receptors as well as steroid hormone dependent growth factors and angiogenic modulators are going to be discussed.

Estrogen Receptor Expression in Prostate Cancer and Premalignant Prostatic Lesions

Estrogens have been implicated in prostatic cancerogenesis and tumor progression. The mechanisms underlying estrogen signaling in human prostate tissue, however, remain poorly understood. Using immunohistochemical and in situ hybridization (ISH) techniques, the present study demonstrates the classical estrogen receptor (ERalpha) in premalignant lesions and prostatic adenocarcinoma through the various stages of the disease. Conversely, the novel characterized ERbeta subtype was undetectable in human prostate tissue. High-grade prostatic intraepithelial neoplasia revealed ERalpha mRNA and protein expression in 28% and 11% of cases evaluated. Focal ER immunoreactivity was detected in a minority of low- to intermediate-grade adenocarcinoma. High-grade (primary Gleason grade 4 and 5) tumors revealed ER protein expression in 43% (62% respectively) of cases. The most significant ERalpha gene expression on mRNA and protein levels was observed in hormone refractory tumors and metastatic lesions, including lymph node and bone metastases. Results of the current study suggest that estrogens can affect prostatic cancerogenesis and neoplastic progression through an ER-mediated process in human prostate tissue.