Epigenetic changes in estrogen receptor β gene in atherosclerotic cardiovascular tissues and in-vitro vascular senescence

Epigenetic changes marked by DNA methylation have been proposed to play a role in age-related disease. We investigated DNA methylation changes in cardiovascular atherosclerotic tissues and in-vitro vascular senescence in the promoter of estrogen receptor β gene, which has essential roles in vascular function. Coronary atherosclerotic tissues showed higher methylation levels (28.7%) than normal appearing arterial (6.7%–10.1%) and venous tissues (18.2%). In comparing estrogen receptor β methylation between plaque and non-plaque regions in ascending aorta, common carotid artery, and femoral artery of two patients, the plaque lesions showed consistently higher methylation levels than non-plaque regions. Passage-dependent increased estrogen receptor β methylation was observed in three of six human aortic endothelial or smooth muscle cell lines cultured in-vitro to vascular senescence. Estrogen receptor β expression in these vascular cell lines was significantly activated by DNA-methyltransferase inhibition. This activity was augmented by histone deacetylase inhibition. These findings provide evidence of epigenetic dysregulation of estrogen receptor β in atherosclerosis and vascular aging. We suggest that focal epigenetic changes in estrogen receptor β contribute to the development of atherosclerosis and vascular aging.     

Association of estrogen receptor beta gene polymorphism with bone mineral density

Chromosomal mapping of the human estrogen receptor beta (ERbeta) gene by fluorescence in situ hybridization (FISH) reveals that ERbeta is located at human chromosome 14, region q23-24.1, where the aberration of DNA copy number in the bone disorders is frequently involved. Then, we investigated the association between dinucleotide (cytosine-adenine; CA) repeat polymorphism located in the flanking region of ERbeta gene and bone mineral density (BMD) in 204 healthy postmenopausal Japanese women. The genotype was classified into "A" through "O" according to the number of the repeats, from 18 to 32. BMD was expressed in Z score (a deviation from the weight-adjusted average BMD of each age using the standard deviation as a unit). When we separate the subjects into two groups bearing at least one I allele (26 CA repeats) and those who did not, the former subjects had significantly higher Z score of L2-4 BMD (mean +/- standard deviation; 0.674 +/- 1.53 vs 0.128 +/- 1.38; P = 0.027). These data suggest that genetic variation at the ERbeta locus may be associated with some determinants for BMD and the possible involvements of this polymorphism in the cause of postmenopausal osteoporosis in Japanese women.     

Expression of estrogen receptor beta is developmentally regulated in reproductive tissues of male and female mice

By the use of ribonuclease protection assay (RPA) combined with immunohistochemical techniques, the expression of estrogen receptor (ER) alpha and ERbeta was mapped in the developing gonads and reproductive tracts of male and female mice from fetal day 14 to postnatal day 26 (PND 26). This study was designed to determine the pattern of expression of both ER subtypes in specific tissue compartments during development. In ovaries, ERalpha mRNA was detected at all ages examined; ERbeta mRNA was seen as early as PND 1, and its expression increased with age. Immunolocalization showed ERbeta in differentiating granulosa cells of the ovary, whereas ERalpha was predominantly seen in interstitial cells. The remainder of the female reproductive tract showed ERalpha mRNA at all ages examined with little or no significant levels of ERbeta, except on PND 1 when a low level of message appeared. In males, ERalpha and ERbeta mRNA were detected in the fetal testis; however, ERbeta gradually increased until PND 5 and subsequently diminished to undetectable levels by PND 26. Immunolocalization showed ERalpha in the interstitial compartment of the testis, whereas ERbeta was seen predominantly in developing spermatogonia. The remainder of the male reproductive tract showed varying amounts of both receptors by RPA and immunostaining throughout development. These studies provide information useful in studying the role of both ER subtypes in normal differentiation, and they provide indications of differential tissue expression during development.     

Lack of functional estrogen receptor beta gene disrupts pubertal male sexual behavior

The estrogen receptor-beta (ERbeta) mediates estrogen action in the female gonads, reproductive tract, and central nervous system. In addition, in rats and mice, gonadotropin-releasing hormone (GnRH-I) neurons coexpress ERbeta. Here we asked if ERbeta plays a role in the onset of puberty and in hypothalamic-pituitary-gonadal (HPG) axis function in male mice. We examined mating behavior, testosterone concentrations, steroid negative feedback on gonadotropins, and GnRH-I function in male ERbeta knockout (ERbetaKO) and wild-type (WT) mice. Peripubertal ERbetaKO males displayed their first ejaculation at a significantly older age than WT littermates. Castrated, adult ERbetaKO mice had significantly higher plasma luteinizing hormone (LH) than WT counterparts. Estradiol (E2) treatment reduced LH and follicle stimulating hormone (FSH) concentrations to an equivalent degree in castrates of both genotypes. In three different measures of the adult GnRH-I system, no genotypic differences were observed. These data show that ERbeta plays an important role in the timing of male sexual behavior at puberty, but does not appear to be involved in adult HPG axis functioning. Furthermore, our data suggest that a primary role of ERbeta may be to regulate ejaculatory behavior.     

雌激素受体p在胃腺癌中表达的研究

目的：检测雌激素受体β（ERβ）在胃组织的存在状况并研究其在人胃腺癌中的作用。方法：使用免疫组化方法,在蛋白水平对配对的原发性胃腺癌患者的癌组织及其癌旁非癌组织的ERB亚型进行检测,采用20例正常胃粘膜作为对照。结果：ERβ蛋白在部分胃腺癌及其癌旁非癌组织表达,但ERβ阳性率及表达模式不同。与配对的非癌组织相比,部分癌组织发生了ERβ表达减少或丢失,而且ERβ表达减少与低分化程度相关（P=0．041）,丢失的ERβ仅见于低分化癌。结论：ERβ可作为识别某些进展期胃腺癌发生发展的标志物,ERβ表达改变在低分化癌中更常见,也提示ERβ阳性胃腺癌可能比ERp表达丢失者预后更好;另外,在非癌组织腺上皮存在E邢的表达提示在正常胃组织中ERB很可能具有一种保护性作用。     

Impaired estrogen sensitivity in bone by inhibiting both estrogen receptor alpha and beta pathways

Although it is well established that estrogen deficiency causes osteoporosis among the postmenopausal women, the involvement of estrogen receptor (ER) in its pathogenesis still remains uncertain. In the present study, we have generated rats harboring a dominant negative ERalpha, which inhibits the actions of not only ERalpha but also recently identified ERbeta. Contrary to our expectation, the bone mineral density (BMD) of the resulting transgenic female rats was maintained at the same level with that of the wild-type littermates when sham-operated. In addition, ovariectomy-induced bone loss was observed almost equally in both groups. Strikingly, however, the BMD of the transgenic female rats, after ovariectomized, remained decreased even if 17beta-estradiol (E(2)) was administrated, whereas, in contrast, the decrease of littermate BMD was completely prevented by E(2). Moreover, bone histomorphometrical analysis of ovariectomized transgenic rats revealed that the higher rates of bone turnover still remained after treatment with E(2). These results demonstrate that the prevention from the ovariectomy-induced bone loss by estrogen is mediated by ER pathways and that the maintenance of BMD before ovariectomy might be compensated by other mechanisms distinct from ERalpha and ERbeta pathways.     

Immunolocalization of estrogen receptor alpha, estrogen receptor beta and androgen receptor in the pre-, peri- and post-pubertal stallion testis

In various species, androgens and estrogens regulate the function of testicular Leydig, Sertoli, peritubular myoid, and germ cells by binding to their respective receptors and eliciting a cellular response. Androgen receptor (AR) is expressed in Sertoli cells, peritubular myoid cells, Leydig cells and perivascular smooth muscle cells in the testis depending on the species, but its presence in germ cells remains controversial. Two different estrogen receptors have been identified, estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ), and their localization and function in testicular cells varies depending on the species, developmental stage of the cell and type of receptor. The localization of AR in an immature and mature stallion has been reported but estrogen receptors have only been reported for the mature stallion. In the present study, the localizations of AR and ERα/ERβ were investigated in pre-pubertal, peri-pubertal and post-pubertal stallions. Testes were collected by routine castration from 21 horses, of light horse breeds (3 months-27 years). Animals were divided into the following age groups: pre-pubertal (3-11 months; n=7), peri-pubertal (12-23 months; n=7) and post-pubertal (2-27 years; n=7). Testicular tissue samples were fixed and embedded, and the presence of AR, ERα and ERβ was investigated by immunohistochemistry (IHC) using procedures previously validated for the horse. Primary antibodies used were rabbit anti-human AR, mouse anti-human ERβ and rabbit anti-mouse ERα. Sections of each region were incubated with normal rabbit serum (NRS; AR and ERα) or mouse IgG (ERβ) instead of primary antibody to generate negative controls. Androgen receptors were localized in Leydig, Sertoli and peritubular myoid cells of all ages. Estrogen receptor alpha was localized in Leydig and germ cells of all ages but only in pre- and peri-pubertal Sertoli cells and post-pubertal peritubular myoid cells. Estrogen receptor beta was localized in Leydig and Sertoli cells of all ages but in only pre-pubertal germ cells and absent in peritubular myoid cells of all ages. Taken together, the data suggest that estrogen regulates steroidogenesis by acting through ERα and ERβ in the Leydig cells and promotes gametogenesis by acting through ERβ in the Sertoli cells and ERα in the germ cells. In contrast androgen receptors are not found in germ cells throughout development and thus are likely to support spermatogenesis by way of a paracrine/autocrine pathway via its receptors in Leydig, Sertoli and peritubular myoid cells.     

Alternative splicing of the estrogen receptor primary transcript normally occurs in estrogen receptor positive tissues and cell lines

Several laboratories have described estrogen receptor mRNA variants created by skipping internal exons. Some of the putative proteins encoded for by these variants have been functionally characterized by transfection analyses. The variant lacking exon 5 would lead, if translated, to a truncated receptor which shows dominant positive transactivation activity in the absence of hormone. It has been postulated that the variant could account for anti-estrogen resistant tumor growth and for expression of the progesterone receptor in estrogen receptor negative tumors. In order to understand the possible role this and other variants may have in the tumorigenesis of mammary tissue we have carried out a thorough analysis of variants expressed in a tumor cell line (MCF-7), in a tumor sample and in a sample of normal breast tissue derived from mammary reduction surgery. We performed rt-PCR analyses followed by hybridization with exon specific oligonucleotide probes. By these means we have detected nine different variants co-expressed in MCF-7 cells and at least the major variants were equally expressed in normal and neoplastic breast tissue. The same is true for the variant lacking exon 5 which, however, resulted to be a variant of low expression in the three samples analyzed. Variant formation appeared to be restricted to the estrogen receptor messenger since several other members of the superfamily of nuclear receptors did not show variant formation. We also have analyzed the effect of the most abundantly expressed variant, the exon 4 lacking variant, on normal estrogen receptor function, on the growth and on the response to estradiol and to tamoxifen of MCF-7 cells. Although over-expressed at high levels this variant has, if any, only marginal effects on the expression of endogenous estrogen regulated genes and on growth and response to the hormone and its antagonist. Although the lack of function of this variant cannot be extrapolated to other variants, their involvement in tumor formation appears rather unlikely since they are also expressed in normal tissue and the single variant is expressed in addition to many others, some of which might have opposing effects. Variant formation is, however, specific for the estrogen receptor and apparently regulated with tissue specificity as our expression analysis in normal mouse tissues shows. Therefore the variants probably have a physiological significance yet to be discovered.     

Epigenetic mechanisms in senescence,immortalisation and cancer

Cancer is controlled not only by genetic events but also by epigenetic events. The active acquisition of epigenetic changes is a poorly understood but very important process in mammalian development, differentiation, and disease. It is well established that epigenetic events are controlled by a specific subgroup of proteins, such as DNA methyltransferases, histone acetylases histone lysine methyltransferases or histone deacetylases, that influence methylation or acetylation patterns to modulate gene expression. We and others have identified S‐adenosylhomocysteine hydrolase in a high‐throughput genetic screen focused on discovering novel genes whose inhibition induces immortalisation of primary cells. Herein, we address the importance of genes involved in epigenetic mechanisms during senescence and how their effects might determine senescence bypass and immortalisation. The ways in which genes that regulate epigenetic mechanisms might modulate senescence/immortalisation and how these pathways could influence cancer development are explored. Overall, epigenetic modifications seem to play a major role in cancer, influencing tumour outcome by interfering with key senescence pathways.