Estrogen receptor beta-selective agonists stimulate calcium oscillations in human and mouse embryonic stem cell-derived neurons.

Estrogens are used extensively to treat hot flashes in menopausal women. Some of the beneficial effects of estrogens in hormone therapy on the brain might be due to nongenomic effects in neurons such as the rapid stimulation of calcium oscillations. Most studies have examined the nongenomic effects of estrogen receptors (ER) in primary neurons or brain slices from the rodent brain. However, these cells can not be maintained continuously in culture because neurons are post-mitotic. Neurons derived from embryonic stem cells could be a potential continuous, cell-based model to study nongenomic actions of estrogens in neurons if they are responsive to estrogens after differentiation. In this study ER-subtype specific estrogens were used to examine the role of ERalpha and ERbeta on calcium oscillations in neurons derived from human (hES) and mouse embryonic stem cells. Unlike the undifferentiated hES cells the differentiated cells expressed neuronal markers, ERbeta, but not ERalpha. The non-selective ER agonist 17beta-estradiol (E(2)) rapidly increased [Ca2+]i oscillations and synchronizations within a few minutes. No change in calcium oscillations was observed with the selective ERalpha agonist 4,4',4''-(4-Propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT). In contrast, the selective ERbeta agonists, 2,3-bis(4-Hydroxyphenyl)-propionitrile (DPN), MF101, and 2-(3-fluoro-4-hydroxyphenyl)-7-vinyl-1,3 benzoxazol-5-ol (ERB-041; WAY-202041) stimulated calcium oscillations similar to E(2). The ERbeta agonists also increased calcium oscillations and phosphorylated PKC, AKT and ERK1/2 in neurons derived from mouse ES cells, which was inhibited by nifedipine demonstrating that ERbeta activates L-type voltage gated calcium channels to regulate neuronal activity. Our results demonstrate that ERbeta signaling regulates nongenomic pathways in neurons derived from ES cells, and suggest that these cells might be useful to study the nongenomic mechanisms of estrogenic compounds.

Organ messenger ribonucleic acid and plasma proteome changes in the adjuvant-induced arthritis model: responses to disease induction and therapy with the estrogen receptor-beta selective agonist ERB-041.

Two receptors [estrogen receptor (ER)alpha and ERbeta] mediate the manifold effects of estrogens throughout the body. Although a clear role has been established for ERalpha in the classical effects of estrogen activity, the physiological role of ERbeta is less well understood. A small-molecule ERbeta selective agonist, ERB-041, has potent antiinflammatory activity in the Lewis rat model of adjuvant-induced arthritis. To characterize the response of target organs and pathways responsible for this antiinflammatory effect, mRNA expression profiling of the spleen, lymph node, and liver was performed, in conjunction with a global analysis of the plasma proteome. We find that the expression of a large number of genes and proteins are altered in the disease model and the majority of these are partially or fully reversed by ERB-041 treatment. Regulated pathways include the acute-phase response, eicosanoid synthesis, fatty acid metabolism, and iron metabolism. In addition, many of the regulated genes and proteins are known to be dysregulated in human rheumatoid arthritis, providing further evidence that the manifestations of the Lewis rat adjuvant-induced arthritis model bear similarity to the human disease.

Determination of rat oral bioavailability of soy-derived phytoestrogens using an automated on-column extraction procedure and electrospray tandem mass spectrometry.

In recent years, consumption of herbal supplements as an alternative to pharmaceutical drug therapy has increased. For example, with the health claims labeling which describes the link between soy-protein and a reduced risk of coronary heart disease (CHD), the consumption of soy and soy-derived phytoestrogens has increased dramatically. That being said, the oral bioavailability of only a few soy phytoestrogens such as Daidzein and Genestein have been previously estimated. In this paper, we present the calculated percent of rat oral bioavailability of five soy-derived phytoestrogens (Genistein, Daidzein, Biochanin A, Coumestrol, and Zearalenone) in male Sprague-Dawley rats. The plasma quantitation required for the bioavailability calculation is performed by using a rapid on-line plasma extraction procedure for the quantitative analysis. To further speed up the analysis the rats were dosed using the 'n-in-one' (cassette) protocol. The rapid on-line extraction/quantitation methodology coupled to the cassette dosing analysis of phytoestrogens is the key point of this paper. The limit of quantitation (LOQ) for each compound was 1-1000 ng/ml with each plasma sample analysis taking less than 2 min. In general the percent oral bioavailability was determined to be between 11 and 28%.

Evaluation of an estrogen receptor-beta agonist in animal models of human disease.

The discovery of a second estrogen receptor (ER), called ERbeta, in 1996 sparked intense interest within the scientific community to discover its role in mediating estrogen action. However, despite more than 6 yr of research into the function of this receptor, its physiological role in mediating estrogen action remains unclear and controversial. We have developed a series of highly selective agonists for ERbeta and have characterized their activity in several clinically relevant rodent models of human disease. The activity of one such compound, ERB-041, is reported here. We conclude from these studies that ERbeta does not mediate the bone-sparing activity of estrogen on the rat skeleton and that it does not affect ovulation or ovariectomy-induced weight gain. In addition, these compounds are nonuterotrophic and nonmammotrophic. However, ERB-041 has a dramatic beneficial effect in the HLA-B27 transgenic rat model of inflammatory bowel disease and the Lewis rat adjuvant-induced arthritis model. Daily oral doses as low as 1 mg/kg reverse the chronic diarrhea of HLA-B27 transgenic rats and dramatically improve histological disease scores in the colon. The same dosing regimen in the therapeutic adjuvant-induced arthritis model reduces joint scores from 12 (maximal inflammation) to 1 over a period of 10 d. Synovitis and Mankin (articular cartilage) histological scores are also significantly lowered (50-75%). These data suggest that one function of ERbeta may be to modulate the immune response, and that ERbeta-selective ligands may be therapeutically useful agents to treat chronic intestinal and joint inflammation.

Constrained phytoestrogens and analogues as ERbeta selective ligands.

A new series of ERbeta (ERbeta) selective ligands has been prepared. One of the compounds 6, structurally related to the phytoestrogen apigenin 4, displays a binding preference for ERbeta over ERalpha of over 40-fold. In addition to its binding selectivity, 6 was able to potently induce metallothionein (an ERbeta specific response in human SAOS-2 cells) while demonstrating low potency in an ERalpha dependant ERE-tk luciferase assay in MCF-7 cells. Such receptor and cell selectivity could make 6 a useful molecular probe for better understanding the role of ERbeta in mammalian physiology.

Characterization of the biological roles of the estrogen receptors, ERalpha and ERbeta, in estrogen target tissues in vivo through the use of an ERalpha-selective ligand.

Estrogens elicit many biomedically important responses in different target tissues, and the respective roles of the two estrogen receptors, ERalpha and ERbeta, in mediating these bioactivities is incompletely understood. In this study, we investigated the activity of an ERalpha-selective agonist ligand, propyl pyrazole triol (PPT), in several rat animal models to define the involvement of ERalpha in these biological responses. In a short-term (4 d) uterotrophic assay, PPT was found to be as efficacious as 17alpha-ethinyl-17beta-estradiol in stimulating uterine weight gain and up-regulating complement 3 gene expression. In a 6-wk chronic model, PPT completely prevented the ovariectomy-induced body weight increase and loss of bone mineral density. It also increased uterine weight and markedly reduced plasma cholesterol levels in these mature animals. PPT was also effective in the brain. It increased progesterone receptor mRNA in the arcuate and ventromedial nuclei of the hypothalamus and prevented experimentally induced hot flushes. Our findings indicate that several physiologically relevant estrogen-induced tissue responses can be effectively evoked via ERalpha alone. By providing an approach that is complementary to that of analyzing the phenotype and response of ER knockout animals, our findings also demonstrate that ER subtype-selective ligands can play a valuable role in enhancing our understanding of how estrogens work through the two ER subtypes.

The ligand binding profiles of estrogen receptors alpha and beta are species dependent.

Estrogens and selective estrogen receptor modulators are used for the treatment and prevention of conditions resulting from menopause. Since estrogens exert their activity by binding to nuclear receptors, there is intense interest in developing new ligands for the two known estrogen receptor subtypes, ER-alpha and ER-beta. Characterization assays used to profile new estrogen receptor ligands often utilize receptors from different species, with the assumption that they behave identically. To test this belief, we have profiled a number of estrogens, other steroids, phytoestrogens and selective estrogen receptor modulators in a solid phase radioligand binding assay using recombinant protein for human, rat, and mouse ER-alpha and ER-beta. Certain compounds show species dependent binding preferences for ER-alpha or ER-beta, leading to differences in receptor subtype selectivity. The amino acids identified by crystallography as lining the ligand binding cavity are the same among the three species, suggesting that as yet unidentified amino acids contribute to the structure of the binding site. We conclude from this analysis that the ability of a compound to selectively bind to a particular ER subtype can be species dependent.