Estrogens are known to regulate the proliferation of breast cancer cells and to alter their cytoarchitectural and phenotypic properties, but the gene networks and pathways by which estrogenic hormones regulate these events are only partially understood. We used global gene expression profiling by Affymetrix GeneChip microarray analysis, with quantitative PCR verification in many cases, to identify patterns and time courses of genes that are either stimulated or inhibited by estradiol (E2) in estrogen receptor (ER)-positive MCF-7 human breast cancer cells. Of the >12,000 genes queried, over 400 showed a robust pattern of regulation, and, notably, the majority (70%) were down-regulated. We observed a general up-regulation of positive proliferation regulators, including survivin, multiple growth factors, genes involved in cell cycle progression, and regulatory factor-receptor loops, and the down-regulation of transcriptional repressors, such as Mad4 and JunB, and of antiproliferative and proapoptotic genes, including B cell translocation gene-1 and -2, cyclin G2, BCL-2 antagonist/killer 1, BCL 2-interacting killer, caspase 9, and TGFbeta family growth inhibitory factors. These together likely contribute to the stimulation of proliferation and the suppression of apoptosis by E2 in these cells. Of interest, E2 appeared to modulate its own activity through the enhanced expression of genes involved in prostaglandin E production and signaling, which could lead to an increase in aromatase expression and E2 production, as well as the decreased expression of several nuclear receptor coactivators that could impact ER activity. Our studies highlight the diverse gene networks and metabolic and cell regulatory pathways through which this hormone operates to achieve its widespread effects on breast cancer cells.

We assessed the preclinical characteristics of a novel, stringently screened selective estrogen receptor modulator, bazedoxifene acetate, including its ability to bind to and activate estrogen receptors and promote increased bone mineral density and bone strength in rats, and the effects impacting the uterine endometrium, breast cancer cell proliferation, and central nervous system-associated vasomotor responses in an animal model. Bazedoxifene bound to estrogen receptor-alpha with an IC50 of 26 nm, an affinity similar to that of raloxifene. Bazedoxifene did not stimulate proliferation of MCF-7 cells but did inhibit 17beta-estradiol-induced proliferation with an IC50 of 0.19 nm. In an immature rat uterine model, bazedoxifene (0.5 and 5.0 mg/kg) was associated with less increase in uterine wet weight than either ethinyl estradiol (10 microg/kg) or raloxifene (0.5 and 5.0 mg/kg). Histological analysis revealed that coadministration of bazedoxifene also appeared to reduce raloxifene-stimulated endometrial luminal epithelial cell and myometrial cell hypertrophy. In ovariectomized rats, bazedoxifene was associated with significant increases in bone mineral density at 6 wk, compared with control, and better compressive strength of bone samples from the L4 vertebrae, compared with samples from ovariectomized animals. In the morphine-addicted rat model of vasomotor activity, bone-sparing doses of bazedoxifene alone were not associated with 17beta-estradiol inhibition of increased vasomotor activity. Bazedoxifene acetate represents a promising new treatment for osteoporosis, with a potential for less uterine and vasomotor effects than selective estrogen receptor modulators currently used in clinical practice. Controlled clinical trial data will be needed to confirm these effects.

Compared with placebo, 24 weeks of daily sc abaloparatide increases BMD of the lumbar spine, femoral neck, and total hip in a dose-dependent fashion. Moreover, the abaloparatide-induced BMD increases at the total hip are greater than with the marketed dose of teriparatide. These results support the further investigation of abaloparatide as an anabolic therapy in postmenopausal osteoporosis.

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.

Estrogen receptors ER␣ and ER␤ are members of the family of nuclear hormone receptors and act as ligandinducible transcriptional factors, which regulate the expression of target genes on binding to cognate response elements. We report here the characterization of steroid receptor coactivator-3 (SRC-3), a coactivator of nuclear receptor transcription that is a member of a family of steroid receptor coactivators that includes SRC-1 and transcription intermediate factor-2. SRC-3 enhanced ER␣ and progesterone receptor-stimulated gene transcription in a ligand-dependent manner, but stimulation of ER␤-mediated transcription was not observed. Protein-protein interaction assays, including real-time interaction analyses with BIAcore, demonstrated that the affinity of the ER␣ interaction with SRC-3 was much higher than that observed for the ER␤ interaction with SRC-3. Mutational analysis suggests a potential interplay between the transactivation function-1 and -2 domains of ER␣ and SRC-3. Furthermore, an intrinsic transactivation function was observed in the C-terminal half of SRC-3. Finally, SRC-3 was differentially expressed in various tissues and, among several tumor cells examined, was most abundant in the nuclear fraction of MCF-7 breast cancer cells. Therefore, SRC-3, a third member of a family of steroid receptor coactivators, has a distinct tissue distribution and intriguing selectivity between ER␣ and ER␤. Estradiol (E 2 )1 exerts numerous biological effects in different tissues through an interaction with the estrogen receptor (ER), a member of the steroid/nuclear hormone receptor superfamily (1, 2). Amino acid sequence analyses, transient transfection studies, and mutational dissections of ER indicate that ER can be subdivided into several functional domains (3). The N-terminal A/B domain contains a transactivation function, referred to as TAF-1. The DNA binding domain, the C region, contains two zinc fingers and is responsible for DNA recognition. The ligand binding domain (LBD) and a second transactivation function, referred to as TAF-2, is located at the C-terminal of ER. On binding to hormone, the receptor undergoes an activation and transformation step. The activated ER interacts with specific estrogen response elements that are located in the promoter region of estrogen-regulated genes and influences the rate of gene transcription. Over the past decade, numerous studies have provided a basic understanding of both the effects of ligand (agonist and antagonist) on the ER and the relationship between the structure and function of the ER (4). Nevertheless, little is known regarding the mechanisms involved in the gene-specific and tissue-selective effects mediated by either estrogens or antiestrogens. Furthermore, the molecular mechanisms by which ligand-activated ER influences the basal transcriptional machinery and regulates target gene transcription are mostly unknown.Recently, a new estrogen receptor, named ER␤, has been isolated from rat prostate and human testis (5, 6). The DNA binding domain of ER␤ is 9...

ABSTRACT

The menopausal transition is associated with decreased ovarian function and concomitant decline in estrogen production, which may result in physiological effects such as hot flashes, reduced bone mass, and altered lipid profile. It is well established that these unfavorable changes are effectively offset with estrogen therapy (ET) or, in women with a uterus, estrogens in combination with a progestin (hormone therapy). Selective estrogen receptor (ER) modulators (SERMs), which exhibit both ER agonist and antagonist activities depending on the target tissue, have been regarded as offering the potential to provide the benefits of ET and hormone therapy with an improved safety and tolerability profile. To date, no SERM alone has demonstrated an ideal benefit-risk profile for menopausal therapy. The tissue-selective estrogen complex, or the pairing of a SERM with estrogens, may provide an optimal blend of ER agonist and antagonist activities. We evaluated the physiological profile of this novel therapeutic paradigm by using various in vivo models to assess uterine, vasomotor, lipid, and skeletal responses to a tissue-selective estrogen complex partnering bazedoxifene with conjugated estrogens (CE). Bazedoxifene at 3.0 mg/kg effectively antagonized CE-induced uterine stimulation without reversing the positive effects of CE on vasomotor instability. When paired with CE, bazedoxifene at 3.0 mg/kg reduced total cholesterol levels by up to 20% compared with CE alone and significantly increased total bone density relative to control. These preclinical findings showed that the appropriate dose combination of bazedoxifene/CE exhibits positive vasomotor, lipid, and skeletal responses with minimal uterine stimulation.

The estrogen receptor (ER) belongs to a superfamily of ligand-inducible transcription factors. Functions of these proteins (dimerization, DNA binding, and interaction with other transcription factors) are modulated by binding of their corresponding ligands. It is, however, controversial whether various ER ligands affect the receptor's ability to bind its specific DNA element (ERE).By using real time interaction analysis we have investigated the kinetics of human (h)ER binding to DNA in the absence and presence of 17␤-estradiol, 17␣-ethynyl estradiol, analogs of tamoxifen, raloxifene, and ICI-182,780. We show that ligand binding dramatically influences the kinetics of hER interaction with specific DNA. We have found that binding of estradiol induces the rapid formation of a relatively unstable ER⅐ERE complex, and binding of ICI-182,780 leads to slow formation (k a is approximately 10 times lower) of a stable receptor-DNA complex (k d is almost 2 orders of magnitude lower). Therefore, binding of estradiol accelerates the frequency of receptor-DNA complex formation more than 50-fold, compared with unliganded ER, and more than 1000-fold compared with ER liganded with ICI-182,780. We hypothesize that a correlation exists between the rate of gene transcription and the frequency of receptor-DNA complex formation. We further show that a good correlation exists between the kinetics of hER-ERE interaction induced by a ligand and its biological effect.Steroid hormones are widely distributed small, lipophilic molecules that participate in intracellular communication and control a wide spectrum of developmental and physiological processes. Their effects are mediated by specific intracellular receptors, a family of proteins that are characterized by a high affinity for the corresponding hormones and an ability to discriminate between structurally closely related ligands. These ligand-inducible receptors can modulate transcription of target genes by virtue of their binding to a specific sequence on DNA in target promoters known as hormone response elements. Although distinct proteins, these receptors are members of a large superfamily of steroid hormone receptors and share many common structural and functional features (1-4).Binding of 17␤-estradiol (E 2 ) 1 to estrogen receptor (ER) is followed by a conformational change, leading to dissociation of the receptor from the complex with the heat shock proteins hsp90 and p59 (5, 6), dimerization (7,8), and activation of DNA binding. After DNA binding the activated receptor can interact with basal transcription factors (9). These interactions are thought to stabilize the preinitiation complex at the promoter, allowing RNA polymerase to initiate transcription (10). Recently a number of transcriptional intermediary factors have been identified that can modify estrogen responsiveness, and several of these proteins interact with the ligand binding domain of the ER in a ligand-dependent manner (11-13). It is obvious that the ligand plays a key role in initiating this cascade of events. ...