Effects of estradiol, estrogen receptor subtype-selective agonists and genistein on glucose metabolism in leptin resistant female Zucker diabetic fatty (ZDF) rats.

The leptin resistant Zucker diabetic fatty (ZDF) rats are hyperphagic and become obese, but whereas the males develop type 2 diabetes mellitus (T2DM), the females remain euglycaemic. As estrogen deficiency is known to increase the risk of developing T2DM, we evaluated the role of ER subtypes alpha and beta in the development of glucose tolerance in leptin resistant ovariectomized (OVX) ZDF rats. At least six rats per group were treated with either vehicle (OVX), 17ß-estradiol (E2), ER subtype-selective agonists (Alpha and Beta), or genistein (Gen) for 17 weeks. At the end of the treatment period a glucose tolerance assay was performed and the metabolic flux of (13)C-glucose for the E2 group was investigated. OVX ZDF rats treated with E2, Alpha, Beta, and Gen tolerated the glucose significantly better than untreated controls. E2 treatment increased absorbance/flux of (13)C-glucose to metabolic relevant tissues such liver, adipose tissue, gastrocnemius, and soleus muscle. Moreover, whereas Alpha treatment markedly increased mRNA expression of GLUT4 in gastrocnemius muscle, Beta treatment resulted in the largest fiber sizes of the soleus muscle. Treatment with Gen increased both the mRNA expression of GLUT 4 and the fiber sizes in the skeletal muscle. In addition, E2 and Alpha treatment decreased food intake and body weight gain. In summary, estrogen-improved glucose absorption is mediated via different molecular mechanisms: while activation of ER alpha seems to stimulate muscular GLUT4 functionality, activation of ER beta results in a hypertrophy of muscle fibers. In addition, selective activation of ER alpha decreased food intake and body weight gain. Our data further indicate that ER subtype-selective agonists and genistein improve systemic glucose tolerance also in the absence of a functional leptin signaling pathway.

Estrogen receptor beta is involved in skeletal muscle hypertrophy induced by the phytoecdysteroid ecdysterone.

SCOPE: The phytoectysteroid ecdysterone (Ecdy) was reported to stimulate protein synthesis and enhance physical performance. The aim of this study was to investigate underlying molecular mechanisms particularly the role of ER beta (ERß). RESULTS: In male rats, Ecdy treatment increased muscle fiber size, serum IGF-1 increased, and corticosteron and 17ß-estradiol (E2) decreased. In differentiated C2C12 myoblastoma cells, treatment with Ecdy, dihydrotestosterone, IGF-1 but also E2 results in hypertrophy. Hypertrophy induced by E2 and Ecdy could be antagonized with an antiestrogen but not by an antiandrogen. In HEK293 cells transfected with ER alpha (ERα) or ERß, Ecdy treatment transactivated a reporter gene. To elucidate the role of ERß in Ecdy-mediated muscle hypertrophy, C2C12 myotubes were treated with ERα (ALPHA) and ERß (BETA) selective ligands. Ecdy and BETA treatment but not ALPHA induced hypertrophy. The effect of Ecdy, E2, and BETA could be antagonized by an ERß-selective antagonist (ANTIBETA). In summary, our results indicate that ERß is involved in the mediation of the anabolic activity of the Ecdy. CONCLUSION: These findings provide new therapeutic perspectives for the treatment of muscle injuries, sarcopenia, and cachectic disease, but also imply that such a substance could be abused for doping purposes.

Molecular effects of ER alpha- and beta-selective agonists on regulation of energy homeostasis in obese female Wistar rats.

The molecular mechanisms underlying the effects of selective ER subtype activation on lipogenesis, adipogenesis, lipid utilization and storage as well as glucose metabolism are currently largely unknown and were analyzed in female OVX Wistar rats on a high-fat diet. Rats received estradiol (E2), ER subtype-selective agonists (Alpha and Beta), and genistein (Gen) for 10 weeks. In adipose tissue, treatment with E2, Alpha, and Beta significantly decreased lipogenic (SREBP-1c, FAS) and adipogenic genes (LPL, PPAR gamma). In liver and skeletal muscle of E2-, Alpha-, Beta-, and Gen-treated animals, lipogenesis and triglyceride accumulation were significantly reduced. Increased hepatic and muscular PPAR gamma mRNA expression was observed in untreated, Beta- and Gen-treated animals, which correlates with increased hepatic glucose uptake. However, only untreated animals showed impaired insulin sensitivity compared to all other groups. Therefore, PPAR gamma up-regulation in untreated animals suggests a compensatory mechanism, probably due to increased triglyceride accumulation in non-adipose tissues. Beta- and Gen-treated animals may benefit from the anabolic potency of ER beta that ameliorates lipid and glucose utilization in muscle. Activation of either ER subtype reduces fat enrichment and improves insulin sensitivity. Depending on the investigated tissue, different molecular pathways seem to be involved.

Granulocytes and vascularization regulate uterine bleeding and tissue remodeling in a mouse menstruation model.

Menstruation-associated disorders negatively interfere with the quality of life of many women. However, mechanisms underlying pathogenesis of menstrual disorders remain poorly investigated up to date. Among others, this is based on a lack of appropriate pre-clinical animal models. We here employ a mouse menstruation model induced by priming mice with gonadal hormones and application of a physical stimulus into the uterus followed by progesterone removal. As in women, these events are accompanied by menstrual-like bleeding and tissue remodeling processes, i.e. disintegration of decidualized endometrium, as well as subsequent repair. We demonstrate that the onset of bleeding coincides with strong upregulation of inflammatory mediators and massive granulocyte influx into the uterus. Uterine granulocytes play a central role in regulating local tissue remodeling since depletion of these cells results in dysregulated expression of matrix modifying enzymes. As described here for the first time, uterine blood loss can be quantified by help of tampon-like cotton pads. Using this novel technique, we reveal that blood loss is strongly reduced upon inhibition of endometrial vascularization and thus, is a key regulator of menstrual bleeding. Taken together, we here identify angiogenesis and infiltrating granulocytes as critical determinants of uterine bleeding and tissue remodeling in a mouse menstruation model. Importantly, our study provides a technical and scientific basis allowing quantification of uterine blood loss in mice and thus, assessment of therapeutic intervention, proving great potential for future use in basic research and drug discovery.

Both estrogen receptor subtypes, ERα and ERß, prevent aldosterone-induced oxidative stress in VSMC via increased NADPH bioavailability.

Activation of vascular mineralocorticoid (MR) or estrogen receptors (ER) exerts opposing effects on vascular remodeling. As we have previously shown, activation of either estrogen receptor subtype, ERα or ERß, is fully sufficient to attenuate vascular remodeling in aldosterone salt-treated rats. To further elucidate the underlying mechanism(s) we tested the hypothesis that ER and MR activation might differentially modulate vascular reactive oxygen species (ROS) generation. In support of this concept, aldosterone increased ROS generation in vascular smooth muscle cells as determined by quantitative dihydroethidium fluorescence microscopy. Co-treatment with the selective ERα agonist 16α-LE2, the selective ERß agonist 8ß-VE2 or the non-selective ER agonist 17ß-estradiol (E2) significantly reduced aldosterone-induced ROS generation. The pure ER antagonist ICI 182,780 completely blocked these salutary effects of E2, 16α-LE2 and 8ß-VE2. Activation of ERα or ERß fully blocked the reduction of intracellular nicotinamide adenine dinucleotide phosphate (NADPH) levels observed in aldosterone treated vascular smooth muscle cells. Intracellular NADPH levels were closely associated with expression and activity of the NADPH generating enzyme glucose-6-phosphate dehydrogenase. In conclusion, estrogens attenuate the detrimental vascular effects of excessive MR activation at least in part by preventing the depletion of intracellular NADPH levels.

Estradiol release kinetics determine tissue response in ovariectomized rats.

Estrogen replacement is an effective therapy of postmenopausal symptoms such as hot flushes, bone loss, and vaginal dryness. Undesired estrogen effects are the stimulation of uterine and mammary gland epithelial cell proliferation as well as hepatic estrogenicity. In this study, we examined the influence of different estradiol release kinetics on tissue responsivity in ovariectomized (OVX) rats. Pulsed release kinetics was achieved by ip or sc administration of estradiol dissolved in physiological saline containing 10% ethanol (EtOH/NaCl) whereas continuous release kinetics was achieved by sc injection of estradiol dissolved in benzylbenzoate/ricinus oil (1+4, vol/vol). Initial 3-d experiments in OVX rats showed that pulsed ip estradiol administration had profoundly reduced stimulatory effects on the uterus and the liver compared with continuous release kinetics. On the other hand, both administration forms prevented severe vaginal atrophy. Based on these results, we compared the effects of pulsed (sc in EtOH/NaCl) vs. continuous (sc in benzylbenzoate/ricinus oil) estradiol release kinetics on bone, uterus, mammary gland, and liver in a 4-month study in OVX rats. Ovariectomy-induced bone loss was prevented by both administration regimes. However, pulsed estradiol resulted in lower uterine weight, reduced induction of hepatic gene expression, and reduced mammary epithelial hyperplasia relative to continuous estradiol exposure. We conclude that organ responsivity is influenced by different hormone release kinetics, a fact that might be exploited to reduce undesired estradiol effects in postmenopausal women.

Impact of estradiol, ER subtype specific agonists and genistein on energy homeostasis in a rat model of nutrition induced obesity.

Estrogens are known to be involved in the control of energy homeostasis. Here we investigated the role of ER alpha and ER beta in a model of nutrition induced obesity. Ovariectomized Wistar rats were fed a high fat diet and received either vehicle, E2, ER subtype selective agonists (Alpha and Beta) or genistein. After 10 weeks, body weight, visceral fat, serum leptin, blood lipids, and in the soleus muscle anabolic markers were determined. Treatment with E2 and Alpha decreased body weight, total cholesterol and VLDL. Visceral fat mass, adipocyte size, and serum leptin were reduced by E2, Alpha and Beta. In the soleus muscle, treatment with E2 and Beta modulated Igf1 and Pax7 gene expression and resulted in larger muscle fibers. Our data indicate that blood lipids are affected via ER alpha, whereas activation of ER beta results in an increase of soleus muscle mass. Adipose tissue homeostasis seems to be affected via both ERs.

Comparative analysis of the uterine and mammary gland effects of progesterone and medroxyprogesterone acetate.

OBJECTIVES: In combined hormone replacement therapy (HRT) progestins are used to inhibit estradiol-activated uterine epithelial cell proliferation. In comparison to estradiol-only therapy, combined HRT leads to enhanced proliferation of mammary epithelial cells. In a quantitative mouse model, we assessed the balance between uterine and undesired mammary gland effects for two progestins that are widely used in HRT, progesterone and medroxyprogesterone acetate. STUDY DESIGN: Mice were ovariectomized and after 14 days they were treated subcutaneously with either vehicle, estradiol (100 ng) or estradiol plus increasing doses of progesterone or medroxyprogesterone acetate for three weeks. MAIN OUTCOME MEASURES: Measures for progestogenic mammary gland activity were stimulation of side-branching and stimulation of epithelial cell proliferation. Progestogenic activity in the uterus was assessed by measuring inhibition of estradiol-activated uterine epithelial cell proliferation. ED(50) and ID(50) values for the distinct readouts were obtained and dissociation factors for uterine versus mammary gland activity were calculated. RESULTS: MPA demonstrated uterine activity and mitogenic activity in the mammary gland at the same doses. In contrast, progesterone showed uterine activity at doses lower than those leading to significant stimulation of epithelial cell proliferation in the mammary gland. CONCLUSIONS: Progestins do not behave the same. Use of the natural hormone progesterone, but not MPA, in combined hormone therapy might offer a safety window between uterine effects and undesired proliferative activity in the mammary gland.

Differential effects of 17beta-estradiol and of synthetic progestins on aldosterone-salt-induced kidney disease.

UNLABELLED: Elevated mineralocorticoid levels and female sex hormones have been shown to confer opposing effects on renal injury, but their combined effects are still unknown. OBJECTIVE: Identify the function of estrogens and of different synthetic progestins on aldosterone salt-mediated renal disease. METHODS: The role of 17beta-estradiol, medroxyprogesterone acetate (MPA), and drospirenone during renal injury was studied in Wistar rats subjected to uni-nephrectomy plus aldosterone salt treatment. RESULTS: Aldo-salt treatment of intact, ovariectomized, and estradiol-treated female rats resulted in remnant kidney hypertrophy without structural damage. Co-treatment with MPA, but not with drospirenone, increased kidney hypertrophy, fluid turnover, sodium retention, and potassium excretion. Medroxyprogesterone acetate also caused glomerular, vascular, tubular, and interstitial lesions that were accompanied by increased blood pressure and enhanced NADPH oxidase (p67phox) and sodium channel (alpha-ENaC) expression. Drospirenone, a progestin with anti-mineralocorticoid function, and spironolactone prevented kidney hypertrophy, hypertension, and sodium retention. Drospirenone and spironolactone also increased renal angiotensin II type 2 receptor expression and relieved aldosterone-induced suppression of serum angiotensin II levels. CONCLUSION: The choice of specific synthetic progestins has profound implications on the development of kidney injury and renal gene expression under conditions of elevated aldosterone serum levels and salt intake.

GPR30 does not mediate estrogenic responses in reproductive organs in mice.

The G protein-coupled receptor Gpr30 (Gper) was recently claimed to bind to estradiol and to activate cytoplasmic signal transduction pathways in response to estradiol. However, there are conflicting data regarding the role of Gpr30 as an estrogen receptor (ER): several laboratories were unable to demonstrate estradiol binding to GPR30 or estradiol-activated signal transduction in Gpr30-expressing cells. To clarify the potential role of Gpr30 as an ER, we generated Gpr30-deficient mice. Although Gpr30 was expressed in all reproductive organs, histopathological analysis did not reveal any abnormalities in these organs in Gpr30-deficient mice. Mutant male and female mice were as fertile as their wild-type littermates, indicating normal function of the hypothalamic-pituitary-gonadal axis. Moreover, we analyzed estrogenic responses in two major estradiol target organs, the uterus and the mammary gland. For that purpose, we examined different readout paradigms such as morphological measures, cellular proliferation, and target gene expression. Our data demonstrate that in vivo Gpr30 is dispensable for the mediation of estradiol effects in reproductive organs. These results are in clear contrast to the phenotype of mice lacking the classic ER alpha (Esr1) or aromatase (Cyp19a1). We conclude that the perception of Gpr30 (based on homology related to peptide receptors) as an ER might be premature and has to be reconsidered.

Neurotrophic estrogens: essential profile and endpoints for drug discovery.

Criteria for the early recognition of selective neurotrophic action are crucial for the discovery of estrogens for supplementation therapy. The comparative characterization of 'tool' compounds in different paradigms demonstrates that estrogen-mediated CNS effects are discernible before the manifestation of changes in primary target organs. Agonist activity at, and recruitment of the coactivator SRC-1 by, the estrogen receptor alpha accurately reflect peripheral, but not neurotrophic, efficacy. Interaction with, and SRC-1 recruitment at, the estrogen receptor beta appears to be an essential prerequisite for pronounced CNS effects. Monitoring of the hypothalamo-pituitary-adrenal axis activity and the differential organ-specific induction of estrogen-responsive proteins are helpful for early delineation of CNS efficacy. Behavioral and antioxidant efficacy are useful confirmatory readouts, with limited roles in lead selection. Finally, an algorithm for the identification of estrogens with a neurotrophic profile can be generated by assigning 'performance grades' in a multifarious test array.

In vivo characterization of estrogen receptor modulators with reduced genomic versus nongenomic activity in vitro.

Estrogen receptor (ER) ligands that are able to prevent postmenopausal bone loss, but have reduced activity in the uterus and the mammary gland might be of great value for hormone therapy. It is well established that the classical ER can activate genomic as well as nongenomic signal transduction pathways. In this study, we analyse the in vivo behaviour of ER ligands that stimulate nongenomic ER effects to the same extent as estradiol, but show clearly reduced activation of genomic ER effects in vitro. Using different readout parameters such as morphological changes, cellular proliferation, and target gene induction, we are able to demonstrate that ER ligands with reduced genomic activity in vitro show a better dissociation of bone versus uterine and mammary gland effects than estradiol that stimulates genomic and nongenomic effects to the same extent. We conclude that pathway-selective ER ligands may represent an interesting option for hormone therapy.

G protein-coupled receptor 30 localizes to the endoplasmic reticulum and is not activated by estradiol.

The classical estrogen receptor (ER) mediates genomic as well as rapid nongenomic estradiol responses. In case of genomic responses, the ER acts as a ligand-dependent transcription factor that regulates gene expression in estrogen target tissues. In contrast, nongenomic effects are initiated at the plasma membrane and lead to rapid activation of cytoplasmic signal transduction pathways. Recently, an orphan G protein-coupled receptor, GPR30, has been claimed to bind to and to signal in response to estradiol. GPR30 therefore might mediate some of the nongenomic estradiol effects. The present study was performed to clarify the controversy about the subcellular localization of GPR30 and to gain insight into the in vivo function of this receptor. In transiently transfected cells as well as cells endogenously expressing GPR30, we confirmed that the receptor localized to the endoplasmic reticulum. However, using radioactive estradiol, we observed only saturable, specific binding to the classical ER but not to GPR30. Estradiol stimulation of cells expressing GPR30 had no impact on intracellular cAMP or calcium levels. To elucidate the physiological role of GPR30, we performed in vivo experiments with estradiol and G1, a compound that has been claimed to act as selective GPR30 agonist. In two classical estrogen target organs, the uterus and the mammary gland, G1 did not show any estrogenic effect. Taken together, we draw the conclusion that GPR30 is still an orphan receptor.

Comparative analysis of the uterine and mammary gland effects of drospirenone and medroxyprogesterone acetate.

The role of progestins in combined hormone therapy is the inhibition of uterine epithelial cell proliferation. The Women's Health Initiative study provided evidence for an increased risk of breast cancer in women treated with conjugated equine estrogens plus the synthetic progestin medroxyprogesterone acetate (MPA), compared with conjugated equine estrogens-only treatment. These findings continue to be discussed, and it remains to be clarified whether the results obtained for MPA in the Women's Health Initiative study are directly applicable to other progestins used in hormone therapy. In this study we compared in a mouse model the effects of the synthetic progestins, MPA, and drospirenone in two major target organs: the uterus and mammary gland. As quantitative measures of progestin activity, we analyzed maintenance of pregnancy, ductal side branching in the mammary gland, and proliferation of mammary and uterine epithelial cells as well as target gene induction in both organs. The outcome of this study is that not all synthetic progestins exhibit the same effects. MPA demonstrated uterine activity and mitogenic activity in the mammary gland at the same doses. In contrast, drospirenone behaved similarly to the natural hormone, progesterone, and exhibited uterine activity at doses lower than those leading to considerable proliferative effects in the mammary gland. We hypothesize that the safety of combined hormone therapy in postmenopausal women may be associated with a dissociation between the uterine and mammary gland activities of the progestin component.

Ligand-dependent activation of ER{beta} lowers blood pressure and attenuates cardiac hypertrophy in ovariectomized spontaneously hypertensive rats.

AIMS: The biological effects of oestrogens are mediated by two different oestrogen receptor (ER) subtypes, ERalpha and ERbeta, which might play different, redundant, or opposing roles in cardiovascular disease. Previously, we have shown that the selective ERalpha agonist 16alpha-LE2 improves vascular relaxation, attenuates cardiac hypertrophy, and increases cardiac output without lowering elevated blood pressure in spontaneously hypertensive rats (SHR). Because ERbeta-deficient mice exhibit elevated blood pressure and since the ERbeta agonist 8beta-VE2 attenuated hypertension in aldosterone-salt-treated rats, we have now tested the hypothesis that the isotype-selective ERbeta agonist 8beta-VE2 might be capable of lowering elevated blood pressure in ovariectomized SHR. METHODS AND RESULTS: Treatment of ovariectomized SHR with 8beta-VE2 for 12 weeks conferred no uterotrophic effects but lowered elevated systolic blood pressure (-38 +/- 5 mmHg, n = 31, P < 0.001 vs. placebo) as well as peripheral vascular resistance (-31.3 +/- 4.6%, P < 0.001 vs. placebo). 8beta-VE2 enhanced aortic ERbeta expression (+75.7 +/- 7.1%, P < 0.01 vs. placebo), improved NO-dependent vasorelaxation, augmented phosphorylation of the vasodilator-stimulated phosphoprotein in isolated aortic rings (P < 0.05 vs. placebo), increased cardiac output (+20.4 +/- 2.5%, P < 0.01 vs. placebo), and attenuated cardiac hypertrophy (-22.2 +/- 3.2%, p < 0.01 vs. placebo). 8beta-VE2, in contrast to oestradiol, did not enhance cardiac alpha-myosin heavy chain expression. CONCLUSION: Ligand-dependent activation of ERbeta confers blood pressure lowering effects in SHR that are superior to those of 17beta-estradiol or the ERalpha agonist 16alpha-LE2 and attenuates cardiac hypertrophy primarily by a reduction of cardiac afterload without promoting uterine growth.

Essential role for estrogen receptor beta in stromal-epithelial regulation of prostatic hyperplasia.

Estrogens, acting via estrogen receptors (ER) alpha and beta, exert direct and indirect actions on prostate growth and differentiation. Previous studies using animal models to determine the role of ERbeta in the prostate have been problematic because the centrally mediated response to estrogen results in reduced androgen levels and prostatic epithelial regression, potentially masking any direct effects via ERbeta. This study overcomes this problem by using the estrogen-deficient aromatase knockout mouse and tissue recombination to provide new insight into estrogen action on prostate growth and pathology. Homo- and heterotypic aromatase knockout tissue recombinants revealed stromal aromatase deficiency induced hyperplasia in normal prostatic epithelium due to disruption of paracrine interaction between stroma and epithelia. Treatment of tissue recombinants with an ERbeta-specific agonist demonstrated that stimulation of ERbeta elicits antiproliferative responses in epithelium that are not influenced by alterations to systemic androgen levels or the activation of ERalpha. Additionally, work performed with intact aromatase knockout mice demonstrated that the administration of an ERbeta-specific agonist ablated preexisting prostatic epithelial hyperplasia, whereas an ERalpha-specific agonist did not. Therefore, failed activation of ERbeta, resulting from local stromal aromatase deficiency, in conjunction with increased androgen levels, results in increased epithelial cell proliferation and prostatic hyperplasia. These data demonstrate essential and beneficial effects of estrogens that are necessary for normal growth of the prostate and distinguishes them from those that adversely alter prostate growth and differentiation. This highlights the potential of selective estrogen-receptor modulators, rather than aromatase inhibitors, for the management of dysregulated prostate growth.

Medroxyprogesterone acetate but not drospirenone ablates the protective function of 17 beta-estradiol in aldosterone salt-treated rats.

Controversial results obtained from human and animal studies on the prevention of heart disease by estrogens and progestins warrant a better understanding of nuclear hormone receptor function and interaction. To address this issue and taking into account that effects of synthetic progestins are not only referable to action through the progesterone receptor but may also be mediated by other steroid receptors, we characterized cardiovascular function and inflammatory gene expression in aldosterone salt-treated rats on long-term administration of 17beta-estradiol, medroxyprogesterone acetate, and drospirenone, a new progestogen exhibiting antimineralocorticoid activity. The complex pattern of cardiovascular injury in ovariectomized Wistar rats induced by chronic aldosterone infusion plus a high-salt diet was significantly attenuated in sham-ovariectomized rats and by coadministration of 17beta-estradiol in ovariectomized animals after 8 weeks of continuous treatment. The beneficial role of 17beta-estradiol on blood pressure, cardiac hypertrophy, vascular osteopontin expression, perivascular fibrosis, and impaired NO-dependent relaxation of isolated aortic rings was completely abrogated by coadministration of medroxyprogesterone acetate. In contrast, drospirenone was either neutral or additive to 17beta-estradiol in protecting against aldosterone salt-induced cardiovascular injury and inflammation. The current results support the hypothesis of complex interactions among estrogen, progesterone, glucocorticoid, androgen, and mineralocorticoid receptor signaling in cardiovascular injury and inflammation. Novel progestins, such as drospirenone, confer superior effects compared with medroxyprogesterone acetate in a model of aldosterone-induced heart disease because of its antimineralocorticoid properties.

Protein-structure-based prediction of animal model suitability for pharmacodynamic studies of subtype-selective estrogens.

Subtype-selective estrogens are of increasing importance as tools used to unravel physiological roles of the estrogen receptors, ERalpha and ERbeta, in various species. Although human ERalpha and ERbeta differ by only two amino acids within the binding pockets, we and others recently succeeded in generating subtype-selective agonists. We have proposed that the selectivity of the steroidal compounds 16alpha-lactone-estradiol (16alpha-LE(2), hERalpha selective) and 8beta-vinyl-estradiol (8beta-VE(2), hERbeta selective) is based on the interaction of certain substituents of these compounds with essentially one amino acid in the respective ER binding pockets. For in vitro and ex vivo pharmacological experiments with these compounds we intended to use bovine tissues available from slaughterhouses in larger quantities. Using homology modeling techniques we determined that the amino acid conferring high hERbeta-selectivity to 8beta-VE(2) is not exchanged between human and bovine ERalpha and bovine ERbeta. Thus, we predicted our steroidal hERbeta-selective compound to exhibit only weak agonistic activity at bERbeta and that bovine tissue is therefore not suited for investigation of ERbeta functions. The situation is presumably identical for pig, sheep, and the common marmoset, whereas rats, mice, and rhesus macaques are appropriate animal models to study pharmacological effects of 8beta-VE(2) in vivo. This prediction was confirmed in transactivation studies assessing estradiol (E(2)) and the two subtype-selective ligands on bovine ERbeta and on a series of hERalpha and hERbeta with mutations in their respective ligand-binding pockets. We have shown that the detailed understanding of the interactions of a compound with its target protein enables the identification of relevant species for pharmacological studies.

Aging reduces the efficacy of estrogen substitution to attenuate cardiac hypertrophy in female spontaneously hypertensive rats.

Clinical trials failed to show a beneficial effect of postmenopausal hormone replacement therapy, whereas experimental studies in young animals reported a protective function of estrogen replacement in cardiovascular disease. Because these diverging results could in part be explained by aging effects, we compared the efficacy of estrogen substitution to modulate cardiac hypertrophy and cardiac gene expression among young (age 3 months) and senescent (age 24 months) spontaneously hypertensive rats (SHRs), which were sham operated or ovariectomized and injected with placebo or identical doses of 17beta-estradiol (E2; 2 microg/kg body weight per day) for 6 weeks (n=10/group). Blood pressure was comparable among sham-operated senescent and young SHRs and not altered by ovariectomy or E2 treatment among young or among senescent rats. Estrogen substitution inhibited uterus atrophy and gain of body weight in young and senescent ovariectomized SHRs, but cardiac hypertrophy was attenuated only in young rats. Cardiac estrogen receptor-alpha expression was lower in intact and in ovariectomized senescent compared with young SHRs and increased with estradiol substitution in aged rats. Plasma estradiol and estrone levels were lower not only in sham-operated but surprisingly also in E2-substituted senescent SHRs and associated with a reduction of hepatic 17beta-hydroxysteroid dehydrogenase type 1 enzyme activity, which converts weak (ie, estrone) into potent estrogens, such as E2. Aging attenuates the antihypertrophic effect of estradiol in female SHRs and is associated with profound alterations in cardiac estrogen receptor-alpha expression and estradiol metabolism. These observations contribute to explain the lower efficiency of estrogen substitution in senescent SHRs.

Identification of estrogen receptor ligands leading to activation of non-genomic signaling pathways while exhibiting only weak transcriptional activity.

Estrogen receptors (ERs) stimulate genomic effects by acting as nuclear transcription factors as well as non-genomic effects by activating distinct cytoplasmic protein kinase cascades. Non-genomic effects have been implicated in numerous cellular processes, such as proliferation, differentiation, apoptosis and vasorelaxation. To exploit non-genomic effects mediated by ERalpha for novel hormone replacement regimens, we screened a focused library of steroid receptor ligands to identify compounds exhibiting properties different from estradiol, i.e. substances that selectively stimulate non-genomic signal transduction pathways while exhibiting low genomic activities. Treatment of breast cancer cells and osteosarcoma cells with estradiol, estren, substance A and substance B led to non-genomic activation of Akt (protein kinase B) and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling cascades mediated by Src (Rous Sarcoma Virus, non-receptor tyrosine kinase) and phosphatidylinositol-3-kinase (PI3K) stimulation. Such compounds leading to prominent Akt/ERK activation but exhibiting only weak genomic properties were applied in vasorelaxation assays, modeling physiological non-genomic ER responses. As expected from PI3K and Src activation data, substances were as effective as estradiol in mediating vasorelaxation. We assume that these pathway-selective estrogen receptor ligands may serve as potent lead structures for novel hormone replacement strategies exhibiting lesser side effects than the existing treatment paradigms.