Drug and Cell Type-Specific Regulation of Genes with Different Classes of Estrogen Receptor β-Selective Agonists

Estrogens produce biological effects by interacting with two estrogen receptors, ERα and ERβ. Drugs that selectively target ERα or ERβ might be safer for conditions that have been traditionally treated with non-selective estrogens. Several synthetic and natural ERβ-selective compounds have been identified. One class of ERβ-selective agonists is represented by ERB-041 (WAY-202041) which binds to ERβ much greater than ERα. A second class of ERβ-selective agonists derived from plants include MF101, nyasol and liquiritigenin that bind similarly to both ERs, but only activate transcription with ERβ. Diarylpropionitrile represents a third class of ERβ-selective compounds because its selectivity is due to a combination of greater binding to ERβ and transcriptional activity. However, it is unclear if these three classes of ERβ-selective compounds produce similar biological activities. The goals of these studies were to determine the relative ERβ selectivity and pattern of gene expression of these three classes of ERβ-selective compounds compared to estradiol (E2), which is a non-selective ER agonist. U2OS cells stably transfected with ERα or ERβ were treated with E2 or the ERβ-selective compounds for 6 h. Microarray data demonstrated that ERB-041, MF101 and liquiritigenin were the most ERβ-selective agonists compared to estradiol, followed by nyasol and then diarylpropionitrile. FRET analysis showed that all compounds induced a similar conformation of ERβ, which is consistent with the finding that most genes regulated by the ERβ-selective compounds were similar to each other and E2. However, there were some classes of genes differentially regulated by the ERβ agonists and E2. Two ERβ-selective compounds, MF101 and liquiritigenin had cell type-specific effects as they regulated different genes in HeLa, Caco-2 and Ishikawa cell lines expressing ERβ. Our gene profiling studies demonstrate that while most of the genes were commonly regulated by ERβ-selective agonists and E2, there were some genes regulated that were distinct from each other and E2, suggesting that different ERβ-selective agonists might produce distinct biological and clinical effects

2', 3', 4'-trihydroxychalcone is an Estrogen Receptor Alpha Coagonist

Estrogens in hormone replacement therapy (HRT) decrease menopausal symptoms, but increase the risks of reproductive cancers. The beneficial effects of estrogen on peripheral tissues and the adverse proliferative effects on the uterus and mammary gland are mediated by ERalpha. Currently HRT is approved only for short-term use. Short-term HRT works for decreasing symptoms associated with menopause, however, long-term usage is needed to prevent subclinical diseases. Because estradiol-bound ERalpha is an agonist in all tissues there is a need for development of more tissue-selective estrogens that can be used for both short and long-term HRT. Chalcones display antiproliferative activity through ERbeta and may have benefits on menopause-induced hot flashes, however activity through ERalpha and their effects on both estradiol gene regulation and physiology are less known. The present study aimed at identifying a chalcone compound which could change the activity of ERalpha in the presence of estradiol as a coagonist, thereby modulating the response of ERalpha on gene regulation and increasing its tissue specificity. After screening a panel of five chalcone compounds for estrogenic activity in cells cotransfected with ERalpha and an ERE upstream of tk-luciferase, 2', 3', 4'-trihydroxychalcone was identified as a unique ERalpha coagonist. 2', 3', 4'-THC displayed no estrogenic activity on its own, but synergized the activation of the ERE in the presence of estradiol. Competitive binding assays with [3H]-estradiol demonstrated that 2', 3', 4'-THC binds to both ERalpha and ERbeta. Estradiol and SERM-induced genes, KRT-19 and NKG2E, were not regulated by 2', 3', 4'-THC alone. Both KRT-19 and NKG2E were synergized with the combination of 2', 3', 4'-THC and estradiol. Tamoxifen and raloxifene induced expression of NKG2E, but did not synergize the expression in the presence of estradiol. The data demonstrates that 2', 3', 4'-THC behaves as a novel coagonist and not a SERM on gene regulation. A unique gene expression profile was induced in U2OSalpha cells treated with a combination of estradiol and 2', 3', 4'-THC for 24 hours with doses that would allow binding of both ligands to ERalpha at the same time. Functional analysis utilizing the binding affinities of estradiol, 2', 3', 4'-THC and another ERalpha binding chalcone, 2, 2', 4'-THC, demonstrated that a heteroligand complex consisting of estradiol and 2', 3', 4'-THC in ERalpha is possible. Despite the synergistic activation of estradiol regulated genes in U2OSalpha cells, the combination of 2', 3', 4'-THC and estradiol did not induce proliferation of MCF-7 cells. In the same cells 2', 3', 4'-THC blocked estradiol-induced G1 to S phase cell cycle transition without blocking proliferative genes regulated by estradiol. In female ovariectomized mice on a soy-free chow diet treated for four weeks (n=5, per group), 2', 3', 4'-THC did not cause uterine proliferation and blocked estradiol-induced proliferation and gene expression. Although 2', 3', 4'-THC blocked estradiol gene expression in uterine tissue, it regulated and modulated estradiol-induced genes in adipose tissue. Because 2', 3', 4'-THC displays unique coagonist activity through ERalpha without causing proliferation, it may be useful for future HRT and expanding our knowledge of ERalpha regulation and ligand interaction

Should We Make More Bone or Not, As Told by Kisspeptin Neurons in the Arcuate Nucleus.

Since its initial discovery in 2002, the neuropeptide Kisspeptin (Kiss1) has been anointed as the master regulator controlling the onset of puberty in males and females. Over the last several years, multiple groups found that Kiss1 signaling is mediated by the 7TM surface receptor GPCR54. Kiss1 mRNA is highly enriched in the basal medial and lateral subregions of the arcuate nucleus (ARC) in the medial basal hypothalamus. Thus, Kiss1ARC neurons reside in a unique anatomical location ideal for sensing and responding to circulating steroid hormones as well as nutrients. Kiss1 expression is highly responsive to fluctuations of the gonadal hormone, estrogen, with nearly 90% of Kiss1ARC neurons expressing the nuclear hormone estrogen receptor alpha (ERa). Here we review recent research that extends the function of Kiss1ARC neurons beyond the regulation of puberty and highlight their emerging, novel roles in controlling energy allocation, behavioral outputs, and sex-dependent bone remodeling in females. Indeed, some of these previously unknown functions for Kiss1 neurons are quite striking as exemplified by the remarkable increase in bone mass after manipulating estrogen signaling in Kiss1ARC neurons. Taken together, we suggest that Kiss1ARC neurons are highly sensitive to nutritional and hormonal cues that dictate energy utilization and reproduction

Should We Make More Bone or Not, As Told by Kisspeptin Neurons in the Arcuate Nucleus.

Since its initial discovery in 2002, the neuropeptide Kisspeptin (Kiss1) has been anointed as the master regulator controlling the onset of puberty in males and females. Over the last several years, multiple groups found that Kiss1 signaling is mediated by the 7TM surface receptor GPCR54. Kiss1 mRNA is highly enriched in the basal medial and lateral subregions of the arcuate nucleus (ARC) in the medial basal hypothalamus. Thus, Kiss1ARC neurons reside in a unique anatomical location ideal for sensing and responding to circulating steroid hormones as well as nutrients. Kiss1 expression is highly responsive to fluctuations of the gonadal hormone, estrogen, with nearly 90% of Kiss1ARC neurons expressing the nuclear hormone estrogen receptor alpha (ERa). Here we review recent research that extends the function of Kiss1ARC neurons beyond the regulation of puberty and highlight their emerging, novel roles in controlling energy allocation, behavioral outputs, and sex-dependent bone remodeling in females. Indeed, some of these previously unknown functions for Kiss1 neurons are quite striking as exemplified by the remarkable increase in bone mass after manipulating estrogen signaling in Kiss1ARC neurons. Taken together, we suggest that Kiss1ARC neurons are highly sensitive to nutritional and hormonal cues that dictate energy utilization and reproduction

Running the Female Power Grid Across Lifespan Through Brain Estrogen Signaling

The role of central estrogen in cognitive, metabolic, and reproductive health has long fascinated the lay public and scientists alike. In the last two decades, insight into estrogen signaling in the brain and its impact on female physiology is beginning to catch up with the vast information already established for its actions on peripheral tissues. Using newer methods to manipulate estrogen signaling in hormone-sensitive brain regions, neuroscientists are now identifying the molecular pathways and neuronal subtypes required for controlling sex-dependent energy allocation. However, the immense cellular complexity of these hormone-sensitive brain regions makes it clear that more research is needed to fully appreciate how estrogen modulates neural circuits to regulate physiological and behavioral end points. Such insight is essential for understanding how natural or drug-induced hormone fluctuations across lifespan affect women's health

2,3,4-Trihydroxychalcone changes estrogen receptor α regulation of genes and breast cancer cell proliferation by a reprogramming mechanism.

BACKGROUND: Menopausal hormone therapy (MHT) is recommended for only five years to treat vasomotor symptoms and vulvovaginal atrophy because of safety concerns with long-term treatment. We investigated the ability of 2,3,4-trihydroxychalcone (2,3,4-THC) to modulate estrogen receptor (ER)-mediated responses in order to find drug candidates that could potentially prevent the adverse effects of long-term MHT treatment. METHODS: Transfection assays, real time-polymerase chain reaction, and microarrays were used to evaluate the effects of 2,3,4-THC on gene regulation. Radioligand binding studies were used to determine if 2,3,4-THC binds to ERα. Cell proliferation was examined in MCF-7 breast cancer cells by using growth curves and flow cytometry. Western blots were used to determine if 2,3,4-THC alters the E2 activation of the MAPK pathway and degradation of ERα. Chromatin immunoprecipitation was used to measure ERα binding to genes. RESULTS: The 2,3,4-THC/E2 combination produced a synergistic activation with ERα on reporter and endogenous genes in human U2OS osteosarcoma cells. Microarrays identified 824 genes that we termed reprogrammed genes because they were not regulated in U2OS-ERα cells unless they were treated with 2,3,4-THC and E2 at the same time. 2,3,4-THC blocked the proliferation of MCF-7 cells by preventing the E2-induced activation of MAPK and c-MYC transcription. The antiproliferative mechanism of 2,3,4-THC differs from selective estrogen receptor modulators (SERMs) because 2,3,4-THC did not bind to the E2 binding site in ERα like SERMs. CONCLUSION: Our study suggests that 2,3,4-THC may represent a new class of ERα modulators that do not act as a direct agonists or antagonists. We consider 2,3,4-THC to be a reprogramming compound, since it alters the activity of ERα on gene regulation and cell proliferation without competing with E2 for binding to ERα. The addition of a reprogramming drug to estrogens in MHT may offer a new strategy to overcome the adverse proliferative effects of estrogen in MHT by reprogramming ERα as opposed to an antagonist mechanism that involves blocking the binding of estrogen to ERα