Effects of Elderberry Juice from Different Genotypes on Oxidative and Inflammatory Responses in Microglial Cells.

Many species of berries are nutritious food and offer health benefits. However, among the different types of berries, information on health effects of American elderberries (Sambucus nigra subsp. canadensis) has been lacking and little is known about whether elderberry consumption can confer neuroprotective effects on the central nervous system. Microglial cells constitute a unique class of immune cells and exhibit characteristic properties to carry out multifunctional duties in the brain. Activation of microglial cells has been implicated in brain injury and in many types of neurodegenerative diseases. Our recent studies demonstrated the ability for endotoxin (lipopolysaccharide, LPS) and interferon gamma (IFNγ) to induce reactive oxygen species (ROS) and nitric oxide (NO) in murine microglial cells (BV-2) through activating NADPH oxidase and the MAPK pathways. In this study, BV-2 microglial cells were used to examine effects of elderberry juice obtained from different genotypes on oxidative and inflammatory responses induced by LPS and IFNγ. Results show that 'Wyldewood' extract demonstrated antioxidant properties by inhibiting IFNγ-induced ROS production and p-ERK1/2 expression. On the other hand, most juice extracts exerted small effects on LPS-induced NO production and some extracts showed an increase in NO production upon stimulation with IFNγ. The disparity of responses on ROS and NO production from different extracts suggests possible presence of unknown endogenous factor(s) in the extract in promoting the IFNγ-induced iNOS synthesis pathway.

Estrogen receptor-alpha deficiency attenuates autoimmune disease in (NZB x NZW)F1 mice.

Estrogens promote lupus in humans and some mouse models of this disease. Nonetheless, little is known about the role of estrogen receptors in lupus pathogenesis. Here, we report that in females on the lupus-prone (NZB x NZW)F(1) background, disruption of estrogen receptor-alpha (ER alpha or Esr1) attenuated glomerulonephritis and increased survival. ER alpha deficiency also retarded development of anti-histone/DNA antibodies, suggesting that ER alpha promotes loss of immunologic tolerance. Furthermore, ER alpha deficiency in (NZB x NZW)F(1) females attenuated the subsequent development of anti-double-stranded DNA (dsDNA) IgG antibodies, which are associated with glomerulonephritis in this model. We provide evidence that ER alpha may promote lupus, at least in part, by inducing interferon-gamma, an estrogen-regulated cytokine that impacts this disease. ER alpha deficiency in (NZB x NZW)F(1) males increased survival and reduced anti-dsDNA antibodies, suggesting that ER alpha also modulates lupus in males. These studies demonstrate that ER alpha, rather than ER beta, plays a major role in regulating autoimmunity in (NZB x NZW)F(1) mice. Furthermore, our results suggest for the first time that ER alpha promotes lupus, at least in part, by impacting the initial loss of tolerance. These data suggest that targeted therapy disrupting ER alpha, most likely within the immune system, may be effective in the prevention and/or treatment of lupus.

Repression of cancer protective genes by 17beta-estradiol: ligand-dependent interaction between human Nrf2 and estrogen receptor alpha.

Repression of cancer-protective phase II enzymes may help explain why estrogen exposure leads to the development of cancer. In an earlier report we described the ability of 17beta-estradiol (E(2)) to repress phase II enzyme activity in vivo. Phase II enzymes are coordinately regulated via the presence of the antioxidant response element (ARE) in their promoter. We wanted to determine if estrogen receptors (ER) repress ARE-dependent gene expression through a mechanism that requires interaction with Nrf2, the transcription factor that regulates ARE-mediated gene transcription. E(2)-bound ERalpha, but not ERbeta, represses ARE-regulated gene expression in the presence of exogenously expressed Nrf2 as well as when the transactivation domain of Nrf2 was fused to a heterologous DNA-binding domain. Deletion of the activation function-2 (AF-2) and the ligand-binding domain of ERalpha result in a constitutive repression of Nrf2-mediated transcription. Finally, E(2)-bound ERalpha co-immunoprecipitates with Nrf2. Repression of Nrf2-mediated transcription by E(2)-bound ERalpha expands our knowledge of E(2)-regulated genes and provides a potential drug-screening target for the development of selective estrogen receptor modulators with a lower risk of causing cancer.

Tissue-specific estrogenic and non-estrogenic effects of a xenoestrogen, nonylphenol.

Alkylphenols perturb the endocrine system and are considered to have weak estrogenic activities. Although it is known that nonylphenol can bind weakly to the estrogen receptor, it is unclear whether all reported effects of nonylphenol are attributable to its estrogen receptor-binding activity. In order to examine whether alkylphenols have similar effects to the natural hormone, estradiol, we used a mouse model to examine the effects of nonylphenol on gene expression and compared it with estradiol. DNA microarray analysis revealed that, in the uterus, most of the genes activated by this alkylphenol at a high dose (50 mg/kg) were also activated by estradiol. At lower doses, nonylphenol (0.5 mg/kg and 5 mg/kg) had little effect on the genes that were activated by estradiol. Thus, we concluded that the effects of nonylphenol at a high dose (50 mg/kg) were very similar to estradiol in uterine tissue. Moreover, since evaluation of estrogenic activity by gene expression levels was comparable with the uterotrophic assay, it indicated that analysis of gene expression profiles can predict the estrogenic activities of chemicals. In contrast to the similar effects of nonylphenol and estradiol observed in the uterus, in the liver, gene expression was more markedly affected by nonylphenol than by estradiol. This indicated that, in the liver, nonylphenol could activate another set of genes that are distinct from estrogen-responsive genes. These results indicated that nonylphenol has very similar effects to estradiol on gene expression in uterine but not in liver tissue, indicating that tissue-specific effects should be considered in order to elucidate the distinct effects of alkylphenols.

Androgen receptor mRNA expression in the bovine ovary.

Previous studies have shown that androgen receptor (AR) is expressed in granulosa cells of healthy, growing ovarian follicles in rats and primates. However, AR expression in the bovine ovary has not been examined. Therefore, a 346-base pair segment of the bovine AR was cloned and sequenced. Using a ribonuclease protection assay, AR expression was detected in total RNA from bovine ovarian cortex. Expression (absence or presence) of AR mRNA was detected by in situ hybridization in bovine ovarian cortex. Follicles (n = 32) were classified as follows: type 1 (1 layer of flattened granulosa cells), type 2 (1-1.5 layers of cuboidal granulosa cells), type 3 (2-3 layers of granulosa cells), type 4 (4-6 layers of cuboidal granulosa cells and formation of thecal layer), and type 5 (>6 layers of cuboidal granulosa cells, defined theca layer, and antrum formation). Frequency of AR mRNA expression increased (P < 0.001) as follicles entered the growing pool. Expression of AR mRNA was absent in type 1 follicles (n = 8), but present in the granulosa cells of 41% of type 2 follicles (n = 12). In types 3-5 follicles, AR mRNA expression was present in granulosa cells of 100% of follicles examined (n = 4, 4, and 4, respectively) and was greater than type 1 follicles (P = 0.002). These data provide evidence of AR mRNA expression in bovine follicles and suggest that AR mRNA increases during early follicle development.

Dietary soy phytoestrogens and ERalpha signalling modulate interferon gamma production in response to bacterial infection.

Diets rich in soy phytoestrogens have many potential health benefits but isoflavones such as genistein may suppress cell mediated immune function. The effect of dietary phytoestrogens on the host response to infection has not been extensively examined. Mice were fed a diet containing soy phytoestrogens and infected with Mycobacterium avium to establish a chronic infection and inflammatory response. As phytoestrogens may act through classical oestrogen receptors (ER), mice deficient in ERalpha signalling and wild type mice were evaluated for a panel of Type 1-associated cytokines (IFNgamma, IL-12 and IL-18) in the spleen. IFNgamma production in the spleen was increased approximately 4-fold in ERalpha-deficient mice fed a casein-based diet over wild type mice fed a casein-based diet (P < 0.05), suggesting a role for ERalpha in suppressing IFNgamma production. IL-18 levels in spleens of wild type mice were decreased compared to ERalpha-deficient mice on a casein diet. Splenic IL-12 and IL-18 levels were not affected in wild type and ERalpha-deficient mice on the phytoestrogen containing diets, with the exception that whole soy increased IL-12 levels in the tissues of ERalpha deficient mice. We conclude that ERalpha and dietary phytoestrogens can influence production of key regulatory cytokines in response to chronic bacterial infection.

In vitro and in vivo regulation of antioxidant response element-dependent gene expression by estrogens.

Understanding estrogen's regulation of phase II detoxification enzymes is important in explaining how estrogen exposure increases the risk of developing certain cancers. Phase II enzymes such as glutathione-S-transferases (GST) and quinone reductase protect against developing chemically induced cancers by metabolizing reactive oxygen species. Phase II enzyme expression is regulated by a cis-acting DNA sequence, the antioxidant response element (ARE). It has previously been reported that several antiestrogens, but not 17beta-estradiol, could regulate ARE-mediated gene transcription. Our goal was to determine whether additional estrogenic compounds could regulate ARE-mediated gene expression both in vitro and in vivo. We discovered that physiological concentrations (10 nm) of 17beta-estradiol repressed GST Ya ARE-dependent gene expression in vitro. Treatment with other endogenous and anti-, xeno-, and phytoestrogens showed that estrogen receptor/ARE signaling is ligand, receptor subtype, and cell type specific. Additionally, GST and quinone reductase activities were significantly lowered in a dose-dependent manner after 17beta-estradiol exposure in the uteri of mice. In conclusion, we have shown that 17beta-estradiol, and other estrogens, down-regulate phase II enzyme activities. We propose estrogen-mediated repression of phase II enzyme activities may increase cellular oxidative DNA damage that ultimately can result in the formation of cancer in some estrogen-responsive tissues.

Similarities and differences in uterine gene expression patterns caused by treatment with physiological and non-physiological estrogens.

Administration of physiological and non-physiological estrogens during pregnancy or after birth is known to have adverse effects on the development of the reproductive tract and other organs. Although it is believed that both estrogens have similar effects on gene expression, this view has not been tested systematically. To compare the effects of physiological (estradiol; E2) and non-physiological (diethylstilbestrol; DES) estrogens, we used DNA microarray analysis to examine the uterine gene expression patterns induced by the two estrogens. Although E2 and DES induced many genes to respond in the same way, different groups of genes showed varying levels of maximal activities to each estrogen, resulting in different dose-response patterns. Thus, each estrogen has a distinct effect on uterine gene expression. The genes were classified into clusters according to their dose-responses to the two estrogens. Of the eight clusters, only two correlated well with the uterotropic effect of different doses of E2. One of these clusters contained genes that were upregulated by E2, which included genes encoding several stress proteins and transcription factors. The other cluster contained genes that were downregulated by E2, including genes related to metabolism, transcription and detoxification processes. The expression of these genes in estrogen receptor-deficient mice was not affected by E2 treatment, indicating that these genes are affected by the E2-bound estrogen receptor. Thus, of the many genes that are affected by estrogen, it was suggested that only a small number are directly involved in the uterotropic effects of estrogen treatment.

Analysis of temporal changes in the expression of estrogen-regulated genes in the uterus.

In order to understand early events caused by estrogen in vivo, temporal uterine gene expression profiles at early stages were examined using DNA microarray analysis. Ovariectomized mice were exposed to 17beta-estradiol and the temporal mRNA expression changes of ten thousand various genes were analyzed. Clustering analysis revealed that there are at least two phases of gene activation during the period up to six hours. One involved immediate-early genes, which included certain transcription factors and growth factors as well as oncogenes. The other involved early-late genes, which included genes related to RNA and protein synthesis. In clusters of down-regulated genes, transcription factors, proteases, apoptosis and cell cycle genes were found. These hormone-inducible genes were not induced in estrogen receptor (ER) alpha knockout mice. Although expression of ERbeta is known in the uterus, these findings indicate the importance of ERalpha in the changes in gene expression in the uterus.

Uterine responsiveness to estradiol and DNA methylation are altered by fetal exposure to diethylstilbestrol and methoxychlor in CD-1 mice: effects of low versus high doses.

We examined the effects on female CD-1 mice of fetal exposure to low doses of the drug diethylstilbestrol (DES) (0.1 microg/kg/day) and the insecticide methoxychlor (MXC) (10 microg/kg/day) as well as 1000-fold higher doses: 100 microg/kg/day DES and 10,000 microg/kg/day MXC. Pregnant females were administered these chemicals on gestation days 12-18. At 7-8 months of age, female offspring were ovariectomized and implanted for 7 days with a Silastic capsule containing estradiol. Relative to controls, females exposed to the 0.1 microg DES dose showed significantly heavier uteri, while females exposed to the 100 microg DES dose showed significantly lighter uteri. Females exposed prenatally to the 10 microg/kg dose of MXC had significantly heavier uteri relative to females exposed to the 10,000 microg/kg dose of MXC, but neither group differed significantly from controls. Liver weight for females exposed to both doses of DES was significantly greater than controls. Using a microarray approach to analyze DNA methylation, an increase in ribosomal DNA (rDNA) methylation was observed. Sequence data and Southern analysis indicate an increase in 18S rDNA and 45S pre-rDNA methylation in uterine samples exposed prenatally to low and high doses of DES. We thus found opposite effects of fetal exposure to a low and a high dose of DES on the uterine response to estradiol (inverted-U dose-response relationship). In contrast, there was a monotonic dose-response relationship found for prenatal DES exposure on both liver weight and ribosomal DNA hypermethylation.

Estrogen receptor specificity for the effects of estrogen in ovariectomized mice.

Estrogen exerts a variety of important physiological effects, which have been suggested to be mediated via the two known estrogen receptors (ERs), alpha and beta. Three-month-old ovariectomized mice, lacking one or both of the two estrogen receptors, were given estrogen subcutaneously (2.3 micro g/mouse per day) and the effects on different estrogen-responsive parameters, including skeletal effects, were studied. We found that estrogen increased the cortical bone dimensions in both wild-type (WT) and double ER knockout (DERKO) mice. DNA microarray analysis was performed to characterize this effect on cortical bone and it identified four genes that were regulated by estrogen in both WT and DERKO mice. The effect of estrogen on cortical bone in DERKO mice might either be due to remaining ERalpha activity or represent an ERalpha/ERbeta-independent effect. Other effects of estrogen, such as increased trabecular bone mineral density, thymic atrophy, fat reduction and increased uterine weight, were mainly ERalpha mediated.

Brain region-specific up-regulation of mouse apolipoprotein E by pharmacological estrogen treatments.

Cerebral apolipoprotein E (apoE) has been implicated in neuronal protection and repair. Due to the variable levels and types of estrogen receptors within different brain regions, the effect of estrogen on apoE and the mechanism of this effect may vary within different regions. Ovariectomized female C57BL/6 mice were treated with pharmacological levels of 17 beta-estradiol or placebo for 5 days, resulting in supraphysiological plasma levels of estradiol in the treated mice. ApoE and glial fibrillary acidic protein (GFAP) levels were measured in the cortex, hippocampus and diencephalon. 17 beta-Estradiol up-regulated apoE but not GFAP in the cortex and diencephalon, whereas in the hippocampus, GFAP and apoE were equally up-regulated. Treatment of estrogen receptor (ER) alpha knockout mice with 17 beta-estradiol or treatment of C57BL/6 mice with 17 alpha-estradiol, a poor estrogen receptor agonist, specifically induced apoE in the cortex, but not in the diencephalon. These results indicate that 17 beta-estradiol effects on apoE are either directly or indirectly mediated by ER alpha in the diencephalon, while the effects in the cortex may be mediated by a non-classical mechanism or by ER beta. Measurement of mRNA levels in estrogen versus placebo-treated wild-type mice indicated that the effect of 17 beta-estradiol on apoE was not associated with changes in apoE mRNA levels.

Estradiol (E2) elicits SRC phosphorylation in the mouse neocortex: the initial event in E2 activation of the MAPK cascade?

In neocortical explants, E2 activates various signaling components of the MAPK cascade, including B-Raf and MAPK kinase-dependent ERK, suggesting a possible role in the differentiative actions of E2 in the brain. To further characterize the signaling pathways activated by E2, we determined whether c-Src, a member of the Src family of nonreceptor tyrosine kinases and an important modulator of both the MAPK cascade and neuronal differentiation, may play a role in E2 signaling. The present studies show for the first time in the brain that E2 elicits phosphorylation of c-Src on three functionally critical tyrosine residues (Y220, Y423, and Y534), and that this phosphorylation occurs despite disruption of ER alpha (in ER knockout mice). PP2, a Src family kinase inhibitor, suppressed not only E2-induced phosphorylation of c-Src, but ERK phosphorylation as well, suggesting that c-Src may be an upstream regulator of E2 signaling. E2-induced phosphorylation of c-Src is associated with increased tyrosine phosphorylation of Shc, increased association of Shc with Grb2, and induction of Ras, but not Rap1, activation. Together, these data provide evidence that E2 activates a novel c-Src-dependent signal transduction pathway in the developing brain.

Expression of the bovine oestrogen receptor-beta (bERbeta) messenger ribonucleic acid (mRNA) during the first ovarian follicular wave and lack of change in the expression of bERbeta mRNA of second wave follicles after LH infusion into cows.

In a previous study, the ERbeta cDNA protein-coding region was utilised to clone bovine ERbeta. The objectives in this study were to examine (1) ERbeta mRNA expression in ovarian follicles throughout the bovine first follicular wave, and (2) effect of LH infusion into cows on bERbeta mRNA expression during the second follicular wave. In experiment 1, heifers (4-5 per time point) were ovariectomized at 12, 24, 36, 48, 60, 72, 84, 96, 144, or 216 h after emergence of the first follicular wave after oestrus. In experiment 2, saline or LH was pulsed hourly (computer-controlled syringe pump) into cows (n = 31; 5-6 per treatment) at wave emergence for 2 or 4 days: wave 1-saline (W1S), wave 2-saline (W2S), or wave 2-LH (25 microg/h; W2LH). Ovaries were removed on day 2 or day 4 after wave emergence. Follicles, 2-19mm in size, were dissected, frozen, and stored at -80 degrees C for in situ hybridisation with two bERbeta cRNA probes. Expression of bERbeta mRNA was localised in granulosa cells of healthy follicles. In experiment 1, bERbeta mRNA expression did not change with time points of the wave showing no association of bERbeta mRNA expression with follicular selection and dominance. However, bERbeta mRNA expression decreased with increase in size of all follicles. Expression of bERbeta mRNA was greater in very small follicles (2-4 mm) than in large (> or = 9 mm) follicles. In experiment 2, expression of bERbeta mRNA in follicles did not differ either between W1S and W2S or between W2S and W2LH. In summary, bERbeta mRNA expression decreased with increasing follicular size. However, neither stage of the wave (selection or dominance), nor pulsatile infusion of LH influenced bERbeta mRNA expression.

Examination of estradiol effects on the rapid estradiol mediated increase in hippocampal synaptic transmission in estrogen receptor alpha knockout mice.

Hippocampal slices from rats exhibit a rapid increase in basal synaptic transmission following 17 beta-estradiol (E(2)) application. In the current study we examined the role of the classic genomic receptor, estrogen receptor alpha (ER alpha), in mediating E(2) effects on synaptic transmission. E(2) (100 pM) increased the extracellular synaptic response in hippocampal slices from gonadectomized male and female mice lacking a functional ER alpha knockout (ER alpha KO) and wild-type (WT) littermates. No sexually dimorphic differences were observed, however, the increase in the field potential was more pronounced in WT mice. ER antagonists did not block E(2) mediated growth of the synaptic response in ER alpha KO mice. The results suggest that the rapid effect of E(2) on synaptic transmission is not mediated by ER alpha, however, ER alpha appears to modulate non-genomic influences on synaptic transmission.

Dominant bovine ovarian follicular cysts express increased levels of messenger RNAs for luteinizing hormone receptor and 3 beta-hydroxysteroid dehydrogenase delta(4),delta(5) isomerase compared to normal dominant follicles.

The objective was to compare ovarian steroids and expression of mRNAs encoding cytochrome P450 side-chain cleavage, cytochrome P450 17 alpha-hydroxylase, cytochrome P450 aromatase, 3 beta-hydroxysteroid dehydrogenase Delta(4),Delta(5) isomerase, LH, and FSH receptors and estrogen receptor-beta in ovaries of cows with dominant and nondominant ovarian follicular cysts and in normal dominant follicles. Estradiol-17 beta, progesterone, and androstenedione concentrations were determined in follicular fluid using specific RIAs. Dominant cysts were larger than young cysts or dominant follicles, whereas nondominant cysts were intermediate. Estradiol-17 beta (ng/ml) and total steroids (ng/follicle) were higher in dominant cysts than in dominant follicles. Expression of LH receptor and 3 beta-hydroxysteroid dehydrogenase mRNAs was higher in granulosa cells of dominant cysts than in dominant follicles. Nondominant cysts had higher follicular concentrations of progesterone, lower estradiol-17 beta concentrations, and lower expression of steroidogenic enzyme, gonadotropin receptor, and estrogen receptor-beta mRNAs than other groups. In summary, increased expression of LH receptor and 3 beta-hydroxysteroid dehydrogenase mRNAs in granulosa and increased follicular estradiol-17 beta concentrations were associated with dominant cysts compared to dominant follicles. Study of cysts at known developmental stages is useful in identifying alterations in follicular steroidogenesis.

Intraovarian actions of oestrogen.

Oestrogen regulates several hypothalamic and pituitary hormones, which in turn control ovarian functions. Oestrogen and its metabolites, such as catecholoestrogens, also have direct effects within the ovary. This review examines the roles of oestrogen in regulating ovarian folliculogenesis, ovulation and corpus luteum formation. Oestrogen promotes follicular development, which culminates in ovulation, by potentiating follicular development, granulosa cell expression of gonadotrophin receptors, steroidogenesis, and gap junction formation by granulosa cells, and by inhibiting granulosa cell apoptosis. In addition, oestrogen may be needed for corpus luteum formation and maintenance. Studies on mutant mice that either lack one or both of the known oestrogen receptors or are unable to synthesize oestrogen support some but not all of these prior inferences of the roles of oestrogen within the ovary. Although these transgenic mice have proved useful in determining some of the intraovarian actions of oestrogen, they present confounding problems, including hormonal imbalances, that hinder interpretation. Transgenic mice with conditional or tissue-directed mutations in their oestrogen receptors are needed to dissect the ovarian actions of oestrogen further. In addition, microarray technologies, combined with specific hormone treatment regimens are likely to provide an attractive, alternative approach to using mutant mice in clarifying the direct actions of oestrogen in the ovaries of other species.

Estrogen receptor- and aromatase-deficient mice provide insight into the roles of estrogen within the ovary and uterus.

Estrogen receptor (ER)- (alpha, beta, and both alpha and beta) and aromatase (Ar) knockout (KO) mice have been created to assess the biological effects of estrogens. This review article discusses the ovarian and uterine phenotypes of these mice. The data obtained have confirmed some older inferences about how the steroid acts, but have also revealed some unexpected aspects of estrogen action. Mol. Reprod. Dev. 59:336-346, 2001.

The role of estrogen and estrogen receptor-alpha in male adipose tissue.

Males and females both express estrogen receptor (ER) in white adipose tissue (WAT), and estrogens appear to play an important role in regulating WAT in females. However, the role of ER in male WAT was unclear. In this review, we describe our work, which used wild type (WT) and ERalpha-knockout (alphaERKO) male and female mice to determine the role of ERalpha in regulating WAT and brown adipose tissue (BAT). There were progressive increases in WAT with advancing age in alphaERKO compared with WT males; weights of various WAT depots in alphaERKO males were increased by more than 100% compared with WT controls during adulthood. Conversely, BAT weight was similar in alphaERKO and WT males at all ages. Adipocyte areas and numbers were also increased in WAT from alphaERKO compared with WT males. Compared with WT controls, alphaERKO females also had increases in WAT. The alphaERKO mice also had insulin resistance and impaired glucose tolerance, similar to humans lacking ERalpha or aromatase. The obesity in alphaERKO males appeared to involve decreased energy expenditure rather than hyperphagia. In summary, ERalpha absence causes adipocyte hyperplasia and hypertrophy in WAT, but not BAT, and is accompanied by insulin resistance and glucose intolerance in both males and females. These results are the first evidence that the estrogen/ERalpha signaling system is critical in female and male WAT deposition, and may have clinical implications.