The rat, mouse and human estrogen receptor (ER) exists as two subtypes, ER alpha and ER beta, which differ in the C-terminal ligand-binding domain and in the N-terminal transactivation domain. In this study, we investigated the estrogenic activity of environmental chemicals and phytoestrogens in competition binding assays with ER alpha or ER beta protein, and in a transient gene expression assay using cells in which an acute estrogenic response is created by cotransfecting cultures with recombinant human ER alpha or ER beta complementary DNA (cDNA) in the presence of an estrogen-dependent reporter plasmid. Saturation ligand-binding analysis of human ER alpha and ER beta protein revealed a single binding component for [3H]-17beta-estradiol (E2) with high affinity [dissociation constant (Kd) = 0.05 - 0.1 nM]. All environmental estrogenic chemicals [polychlorinated hydroxybiphenyls, dichlorodiphenyltrichloroethane (DDT) and derivatives, alkylphenols, bisphenol A, methoxychlor and chlordecone] compete with E2 for binding to both ER subtypes with a similar preference and degree. In most instances the relative binding affinities (RBA) are at least 1000-fold lower than that of E2. Some phytoestrogens such as coumestrol, genistein, apigenin, naringenin, and kaempferol compete stronger with E2 for binding to ER beta than to ER alpha. Estrogenic chemicals, as for instance nonylphenol, bisphenol A, o, p'-DDT and 2',4',6'-trichloro-4-biphenylol stimulate the transcriptional activity of ER alpha and ER beta at concentrations of 100-1000 nM. Phytoestrogens, including genistein, coumestrol and zearalenone stimulate the transcriptional activity of both ER subtypes at concentrations of 1-10 nM. The ranking of the estrogenic potency of phytoestrogens for both ER subtypes in the transactivation assay is different; that is, E2 >> zearalenone = coumestrol > genistein > daidzein > apigenin = phloretin > biochanin A = kaempferol = naringenin > formononetin = ipriflavone = quercetin = chrysin for ER alpha and E2 >> genistein = coumestrol > zearalenone > daidzein > biochanin A = apigenin = kaempferol = naringenin > phloretin = quercetin = ipriflavone = formononetin = chrysin for ER beta. Antiestrogenic activity of the phytoestrogens could not be detected, except for zearalenone which is a full agonist for ER alpha and a mixed agonist-antagonist for ER beta. In summary, while the estrogenic potency of industrial-derived estrogenic chemicals is very limited, the estrogenic potency of phytoestrogens is significant, especially for ER beta, and they may trigger many of the biological responses that are evoked by the physiological estrogens.

/pdf/interaction-of-estrogenic-chemicals-and-phytoestrogens-with-j0qbzn4u7e.pdf

Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor beta.

https://www.medicinalgenomics.com/wp-content/uploads/2013/01/Resveratrol-rescues-mito-function-through-sirtuin-and-PGC-1.pdf

Resveratrol improves mitochondrial function and protects against metabolic disease by activating SIRT1 and PGC-1alpha.

The Structural Basis of Estrogen Receptor/Coactivator Recognition and the Antagonism of This Interaction by Tamoxifen

The ability to parse tumors into subsets based on biomarker expression has many clinical applications; however, there is no global way to visualize the best cut-points for creating such divisions. We have developed a graphical method, the X-tile plot that illustrates the presence of substantial tumor subpopulations and shows the robustness of the relationship between a biomarker and outcome by construction of a two dimensional projection of every possible subpopulation. We validate X-tile plots by examining the expression of several established prognostic markers (human epidermal growth factor receptor-2, estrogen receptor, p53 expression, patient age, tumor size, and node number) in cohorts of breast cancer patients and show how X-tile plots of each marker predict population subsets rooted in the known biology of their expression.

https://clincancerres.aacrjournals.org/content/clincanres/10/21/7252.full.pdf

X-tile: a new bio-informatics tool for biomarker assessment and outcome-based cut-point optimization

The adult skeleton regenerates by temporary cellular structures that comprise teams of juxtaposed osteoclasts and osteoblasts and replace periodically old bone with new. A considerable body of evidence accumulated during the last decade has shown that the rate of genesis of these two highly specialized cell types, as well as the prevalence of their apoptosis, is essential for the maintenance of bone homeostasis; and that common metabolic bone disorders such as osteoporosis result largely from a derangement in the birth or death of these cells. The purpose of this article is 3-fold: 1) to review the role and the molecular mechanism of action of regulatory molecules, such as cytokines and hormones, in osteoclast and osteoblast birth and apoptosis; 2) to review the evidence for the contribution of changes in bone cell birth or death to the pathogenesis of the most common forms of osteoporosis; and 3) to highlight the implications of bone cell birth and death for a better understanding of the mechanism of action and efficacy of present and future pharmacotherapeutic agents for osteoporosis.

/pdf/birth-and-death-of-bone-cells-basic-regulatory-mechanisms-3uf3t3e9wv.pdf

Birth and death of bone cells: basic regulatory mechanisms and implications for the pathogenesis and treatment of osteoporosis.

Estrogen regulates a plethora of functionally dissimilar processes in a broad range of tissues. Recent progress in the study of the molecular mechanism of action of estrogen(s) has revealed why different cells can respond to the same hormone in a different manner. Three of these findings are of particular importance: (i) There are two genetically and functionally distinct estrogen receptors that have distinct expression patterns in vivo; (ii) the positive and negative transcriptional activities of these receptors require them to engage transcription cofactors (coactivators or corepressors) in target cells; and (iii) not all cofactors are functionally equivalent, nor are they expressed in the same manner in all cells. Thus, although the estrogen receptor is required for a cell to respond to an estrogenic stimulus, the nature and extent of that response are determined by the proteins, pathways, and processes with which the receptor interacts.

https://science.sciencemag.org/content/sci/296/5573/1642.full.pdf

Connections and Regulation of the Human Estrogen Receptor

Estrogen receptor (ER) modulators produce distinct tissue-specific biological effects, but within the confines of the established models of ER action it is difficult to understand why. Previous studies have suggested that there might be a relationship between ER structure and activity. Different ER modulators may induce conformational changes in the receptor that result in a specific biological activity. To investigate the possibility of modulator-specific conformational changes, we have applied affinity selection of peptides to identify binding surfaces that are exposed on the apo-ERs α and β and on each receptor complexed with estradiol or 4-OH tamoxifen. These peptides are sensitive probes of receptor conformation. We show here that ER ligands, known to produce distinct biological effects, induce distinct conformational changes in the receptors, providing a strong correlation between ER conformation and biological activity. Furthermore, the ability of some of the peptides to discriminate between different ER α and ER β ligand complexes suggests that the biological effects of ER agonists and antagonists acting through these receptors are likely to be different.

Estrogen receptor (ER) modulators each induce distinct conformational changes in ER alpha and ER beta

Estrogen receptor α transcriptional activity is regulated by distinct conformational states that are the result of ligand binding. Phage display was used to identify peptides that interact specifically with either estradiol- or tamoxifen-activated estrogen receptor α. When these peptides were coexpressed with estrogen receptor α in cells, they functioned as ligand-specific antagonists, indicating that estradiol-agonist and tamoxifen–partial agonist activities do not occur by the same mechanism. The ability to regulate estrogen receptor α transcriptional activity by targeting sites outside of the ligand-binding pocket has implications for the development of estrogen receptor α antagonists for the treatment of tamoxifen-refractory breast cancers.

https://science.sciencemag.org/content/sci/285/5428/744.full.pdf

Peptide Antagonists of the Human Estrogen Receptor

Recruitment of transcriptional coactivators following ligand activation is a critical step in nuclear receptor-mediated target gene expression. Upon binding an agonist, the receptor undergoes a conformational change which facilitates the formation of a specific coactivator binding pocket within the carboxyl terminus of the receptor. This permits the alpha-helical LXXLL motif within some coactivators to interact with the nuclear receptors. Until recently, the LXXLL motif was thought to function solely as a docking module; however, it now appears that sequences flanking the core motif may play a role in determining receptor selectivity. To address this issue, we used a combinatorial phage display approach to evaluate the role of flanking sequences in influencing these interactions. We sampled more than 10(8) variations of the core LXXLL motif with estradiol-activated estrogen receptor alpha (ERalpha) as a target and found three different classes of peptides. All of these peptides interacted with ERalpha in an agonist-dependent manner and disrupted ERalpha-mediated transcriptional activity when introduced into target cells. Using a series of ERalpha-mutants, we found that these three classes of peptides showed different interaction patterns from each other, suggesting that not all LXXLL motifs are the same and that receptor binding selectivity can be achieved by altering sequences flanking the LXXLL core motif. Most notable in this regard was the discovery of a peptide which, when overexpressed in cells, selectively disrupted ERbeta- but not ERalpha-mediated reporter gene expression. This novel ERbeta-specific antagonist may be useful in identifying and characterizing the ERbeta-regulated process in estradiol-responsive cells. In conclusion, using a combinatorial approach to define cofactor-receptor interactions, we have clearly been able to demonstrate that not all LXXLL motifs are functionally equivalent, a finding which suggests that it may be possible to target receptor-LXXLL interactions to develop receptor-specific antagonists.

Dissection of the LXXLL Nuclear Receptor-Coactivator Interaction Motif Using Combinatorial Peptide Libraries: Discovery of Peptide Antagonists of Estrogen Receptors α and β

Antiestrogens such as tamoxifen are one of the most effective methods of treating estrogen receptor (ERa) positive breast cancers; however, the effectiveness of this therapy is limited by the almost universal development of resistance to the drug If antiestrogens are recognized differently by the cell as it has been suggested, then in disease conditions where tamoxifen fails to function effectively, a mechanistically different antiestrogen might yield successful results Although many antiestrogens have been developed, a direct comparison of their mechanisms of action is lacking, thus limiting their utility Therefore, to determine if there are mechanistic differences among available antiestrogens, we have carried out a comprehensive analysis of the molecular mechanisms of action of 4-hydroxy-tamoxifen (4OHT), idoxifene, raloxifene, GW7604, and ICI 182,780 Using a novel set of peptides that recognize different surfaces on ERa ,w e have found that following binding to ERa, each ligand induces a distinct ERa-ligand conformation Furthermore, transcriptional assays indicate that each ERa-ligand complex is recognized distinctly by the transcription machinery, and consequently, antiestrogens vary in their ability to inhibit estradiol- and 4OHT-mediated activities Relative binding assays have shown that the affinity of these ligands for ERa is not always representative of their inhibitory activity Using this assay, we have also shown that the pharmacology of each antiestrogen is influenced differently by hormone binding proteins Furthermore, GW7604, like ICI 182,780, but unlike the other antiestrogens evaluated, decreases the stability of the receptor Overall, our results indicate that there are clear mechanistic distinctions among each of the antiestrogens studied However, GW7604 and ICI 182,780 differ more significantly from tamoxifen than idoxifene and raloxifene These data, which reveal differences among antiestrogens, should assist in the selection of compounds for the clinical regulation of ERa function (Endocrinology 140: 5828 ‐5840, 1999)

/pdf/comparative-analyses-of-mechanistic-differences-among-26om6psqp2.pdf

John D. Norris

Papers

Connections and Regulation of the Human Estrogen Receptor

Estrogen receptor (ER) modulators each induce distinct conformational changes in ER alpha and ER beta

Peptide Antagonists of the Human Estrogen Receptor

Dissection of the LXXLL Nuclear Receptor-Coactivator Interaction Motif Using Combinatorial Peptide Libraries: Discovery of Peptide Antagonists of Estrogen Receptors α and β

Comparative analyses of mechanistic differences among antiestrogens.