High-Fat Diet Obesity Associated With Insulin Resistance Increases Cell Proliferation, Estrogen Receptor, and PI3K Proteins in Rat Ventral Prostate

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Polymorphisms of estrogen receptor-α gene in Brazilian women with high breast density after menopause

The association of genetic polymorphism in the estrogen receptor alpha (ERα) gene and risk for diseases including breast cancer (BC) has been the subject of great interest. Objective: Checking on women with high breast density after menopause, the frequency of the Pvull and Xbal polymorphisms of the ERα gene and the correlation between them and the known risk factors for breast cancer. Method: Observational study with 308 women between 45 and 65 years old with high breast density, without hormonal therapy, menstruation for a year or more, breast and ovarian cancer history. It was characterized in clinical history and physical examination: menarche, menopause, parity, family history of BC, smoking, alcohol intake and body mass index. Results: The allelic and genotypic frequencies for ERα-Pvull and Xbal: p=43.99%; p=56.01%; pp=32.14%; Pp=47.73% and PP=20.13%; X=41.56%; x=58.44%; xx=33.44%; Xx=50.00% and XX=16.56%, respectively. The most frequent risk factors for BC: menarche before 12 years old (35.38%), nulliparity or first child after 28 years old (41.66%), family history of BC (19.16%) and overweight/obesity (62.01%). Conclusion: Allelic and genotypic distribution similar to literature. The risk factors for BC were more prevalent in women with high breast density but without significant associations with these polymorphisms. © 2013 Informa UK Ltd. All rights reserved.

Differential expression of aromatase, estrogen receptor alpha and 17 beta-HSD associated with the processes of total testicular regression and recrudescence in the bat Myotis nigricans (Chiroptera: Vespertilionidae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

High-Fat Diet Obesity Associated With Insulin Resistance Increases Cell Proliferation, Estrogen Receptor, and PI3K Proteins in Rat Ventral Prostate

In this study, we evaluated the effects of obesity and insulin resistance induced by a high-fat diet on prostate morphophysiology, focusing on cell proliferation, expression of androgen (AR) and estrogen receptors (ER) and proteins of the insulin signaling pathway. Adult male Wistar rats were fed a high-fat diet (20% fat) for 15 weeks, whereas control animals received a balanced diet (4% fat). Both groups were then divided and treated for 2 weeks with 1 mg/kg body weight/day of the aromatase inhibitor letrozole or vehicle only. The ventral prostate was analyzed with immunohistochemical, histopathological, stereological, and Western blotting methods. Obese rats showed insulin resistance, hyperinsulinemia, and reduced plasma testosterone levels. The incidence of prostatic intraepithelial neoplasia (PIN) was 2.7 times higher in obese rats and affected 0.4% of the gland compared with 0.1% PIN areas found in control rats. Obesity doubled cell proliferation in both prostate epithelium and stroma. AR content decreased in the prostate of obese rats and estrogen receptor beta (ER beta) increased in this group. Protein levels of insulin receptor substrate 1 and protein kinase B diminished in the obese group, whereas phosphatidylinositol 3-kinase (PI3K) increased significantly. Most structural changes observed in the prostate of obese rats normalized after letrozole treatment, except for increased stromal cell proliferation and ER beta expression, which might be associated with insulin resistance. This experimental model of obesity and insulin resistance induced by a high-fat diet increases cell proliferation in rat prostate. Such alterations are associated with decreased levels of AR and increased ER beta and PI3K proteins. This change can facilitate the establishment of proliferative lesions in rat prostate.

Regulation of estrogen receptor transcriptional enhancement by the cyclin A/Cdk2 complex

We have found that ectopic expression of cyclin A increases hormone-dependent and hormone-independent transcriptional activation by the estrogen receptor in vivo in a number of cell lines, including HeLa cells, U-2 OS osteosarcoma cells and Hs 578Bst breast epithelial cells. This effect can be further enhanced in HeLa cells by the concurrent expression of the cyclin-dependent kinase activator, cyclin H, and cdk7, and abolished by expression of the cdk inhibitor, p27KIP1, or by the expression of a dominant negative catalytically inactive cdk2 mutant. ER is phosphorylated between amino acids 82 and 121 in vitro by the cyclin A/cdk2 complex and incorporation of phosphate into ER is stimulated by ectopic expression of cyclin A in vivo. Together, these results strongly suggest a direct role for the cyclin A/cdk2 complex in phosphorylating ER and regulating its transcriptional activity.

Responses in the brain of estrogen receptor α-disrupted mice

These studies sought to determine if neurons in the estrogen receptor-α knockout (ERαKO) mouse brain concentrated 16α-[125I]iodo-11β-methoxy-17β-estradiol (125I-estrogen), and if so, whether estrogen binding augmented the expression of progesterone receptor (PR) mRNA. Mice were injected with 125I-estrogen and cryostat sections thaw mounted onto emulsion-coated slides. After 30–90 days of exposure, cells with a nuclear uptake and retention of 125I-estrogen were observed in a number of ERαKO mouse brain regions including the preoptic nucleus and arcuate nucleus of the hypothalamus, bed nucleus of the stria terminalis, and amygdala, although the number of labeled cells and intensity of nuclear concentration was markedly attenuated when compared with wild-type littermates. Competition studies with excess 17β-estradiol, diethylstilbestrol, or moxestrol, but not with R5020 or dihydrotestosterone, prevented the nuclear concentration of 125I-estrogen. To determine if the low level of estrogen binding was capable of regulating gene expression, in situ hybridization was used to evaluate PR mRNA in the brain. ERαKO and wild-type mice were ovariectomized and treated with vehicle or 17β-estradiol, and brains were sectioned and hybridized with a PR cRNA probe. Analysis of hybridization signal revealed a similar, low level of PR mRNA in ovariectomized wild-type and homozygous mice, and a marked increase in expression after treatment of ovariectomized animals with 17β-estradiol, with the level of hybridization signal being significantly higher in wild-type animals when compared with ERαKO mice. The results demonstrate that estrogen binds in the ERαKO brain and is capable of modulating PR gene expression, thus supporting the presence and functionality of a nonclassical estrogen receptor.

Estrogen inhibits the vascular injury response in estrogen receptor β-deficient female mice

The protective effects of estrogen in the cardiovascular system result from both systemic effects and direct actions of the hormone on the vasculature. Two estrogen receptors have been identified, ERα and ERβ. We demonstrated previously that estrogen inhibits the response to vascular injury in both wild-type and ERα-deficient mice, and that ERβ is expressed in the blood vessels of each, suggesting a role for ERβ in the vascular protective effects of estrogen. In the present study, we examined the effect of estrogen administration on mouse carotid arterial injury in ERβ-deficient mice. Surprisingly, in ovariectomized female wild-type and ERβ knockout mice, 17β-estradiol markedly and equally inhibited the increase in vascular medial area and the proliferation of vascular smooth muscle cells after vascular injury. These data demonstrate that ERβ is not required for estrogen-mediated inhibition of the response to vascular injury, and suggest that either of the two known estrogen receptors is sufficient to protect against vascular injury, or that another unidentified estrogen receptor mediates the vascular protective effects of estrogen.

Estrogen receptor specificity in the regulation of skeletal growth and maturation in male mice

Androgens may regulate the male skeleton directly through a stimulation of androgen receptors or indirectly through aromatization of androgens into estrogen and, thereafter, through stimulation of estrogen receptors (ERs). The relative importance of ER subtypes in the regulation of the male skeleton was studied in ERα-knockout (ERKO), ERβ-knockout (BERKO), and double ERα/β-knockout (DERKO) mice. ERKO and DERKO, but not BERKO, demonstrated decreased longitudinal as well as radial skeletal growth associated with decreased serum levels of insulin-like growth factor I. Therefore, ERα, but not ERβ, mediates important effects of estrogen in the skeleton of male mice during growth and maturation.

Tracking the estrogen receptor in neurons: Implications for estrogen-induced synapse formation

Estrogens (E) and progestins regulate synaptogenesis in the CA1 region of the dorsal hippocampus during the estrous cycle of the female rat, and the functional consequences include changes in neurotransmission and memory. Synapse formation has been demonstrated by using the Golgi technique, dye filling of cells, electron microscopy, and radioimmunocytochemistry. N-methyl-d-aspartate (NMDA) receptor activation is required, and inhibitory interneurons play a pivotal role as they express nuclear estrogen receptor alpha (ERα) and show E-induced decreases of GABAergic activity. Although global decreases in inhibitory tone may be important, a more local role for E in CA1 neurons seems likely. The rat hippocampus expresses both ERα and ERβ mRNA. At the light microscopic level, autoradiography shows cell nuclear [3H]estrogen and [125I]estrogen uptake according to a distribution that primarily reflects the localization of ERα-immunoreactive interneurons in the hippocampus. However, recent ultrastructural studies have revealed extranuclear ERα immunoreactivity (IR) within select dendritic spines on hippocampal principal cells, axon terminals, and glial processes, localizations that would not be detectable by using standard light microscopic methods. Based on recent studies showing that both types of ER are expressed in a form that activates second messenger systems, these findings support a testable model in which local, non-genomic regulation by estrogen participates along with genomic actions of estrogens in the regulation of synapse formation.

CTCF modulates Estrogen Receptor function through specific chromatin and nuclear matrix interactions

Enhancer regions and transcription start sites of estrogen-target regulated genes are connected by means of Estrogen Receptor long-range chromatin interactions. Yet, the complete molecular mechanisms controlling the transcriptional output of engaged enhancers and subsequent activation of coding genes remain elusive. Here, we report that CTCF binding to enhancer RNAs is enriched when breast cancer cells are stimulated with estrogen. CTCF binding to enhancer regions results in modulation of estrogen-induced gene transcription by preventing Estrogen Receptor chromatin binding and by hindering the formation of additional enhancer-promoter ER looping. Furthermore, the depletion of CTCF facilitates the expression of target genes associated with cell division and increases the rate of breast cancer cell proliferation. We have also uncovered a genomic network connecting loci enriched in cell cycle regulator genes to nuclear lamina that mediates the CTCF function. The nuclear lamina and chromatin interactions are regulated by estrogen-ER. We have observed that the chromatin loops formed when cells are treated with estrogen establish contacts with the nuclear lamina. Once there, the portion of CTCF associated with the nuclear lamina interacts with enhancer regions, limiting the formation of ER loops and the induction of genes present in the loop. Collectively, our results reveal an important, unanticipated interplay between CTCF and nuclear lamina to control the transcription of ER target genes, which has great implications in the rate of growth of breast cancer cells.