Genetic and pharmacologic strategies to determine the function of estrogen receptor α and estrogen receptor β in cardiovascular system

Background: The biological functions of estrogens extend beyond the female and male reproductive tract, affecting the cardiovascular and renal systems. Traditional views on the role of postmenopausal hormone therapy (HT) in protecting against heart disease, which were challenged by clinical end point studies that found adverse effects of combined HT, are now being replaced by more differentiated concepts suggesting a beneficial role of early and unopposed HT that does not include a progestin.Objective: We reviewed recent insights, concepts, and research results on the biology of both estrogen receptor (ER) subtypes, ERα and ERβ, in cardiac and vascular tissues. Knowledge of these ER subtypes is crucial to understanding gender and estrogen effects and to developing novel, exciting strategies that may have a profound clinical impact.Methods: This review focuses on in vivo studies and includes data presented at the August 2007 meeting of the American Physiological Society as well as data from a search of the MEDLINE and Ovid databases from January 1986 to November 2007. Search results were restricted to English-language publications, using the following search terms: estrogen, estrogen receptor α, estrogen receptor β, estrogen receptor α agonist, estrogen receptor α antagonist, estrogen receptor β agonist, estrogen receptor β antagonist, PPT, DPN, heart, vasculature, ERKO mice, BERKO mice, transgenic mice, and knockout mice.Results: Genetic mouse models and pharmacologic studies that employed selective as well as nonselective ER agonists support the concept that both ER subtypes confer protective effects in experimental models of human heart disease, including hypertension, cardiac hypertrophy, and chronic heart failure.Conclusions: Genetic models and novel ligands hold the promise of further improving our understanding of estrogen action in multiple tissues and organs. These efforts will ultimately enhance the safety and efficacy of HT and may also result in new applications for synthetic female sex hormone analogues.     

Evidence for new C-terminally truncated variants of α- and β-tubulins

Cellular α-tubulin can bear various carboxy-terminal sequences: full-length tubulin arising from gene neosynthesis is tyrosinated, and two truncated variants, corresponding to detyrosinated and Δ2 α‑tubulin, result from the sequential cleavage of one or two C-terminal residues, respectively. Here, by using a novel antibody named 3EG that is highly specific to the –EEEG C-terminal sequence, we demonstrate the occurrence in neuronal tissues of a new αΔ3‑tubulin variant corresponding to α1A/B‑tubulin deleted of its last three residues (EEY). αΔ3‑tubulin has a specific distribution pattern: its quantity in the brain is similar to that of αΔ2-tubulin around birth but is much lower in adult tissue. This truncated α1A/B-tubulin variant can be generated from αΔ2-tubulin by the deglutamylases CCP1, CCP4, CCP5, and CCP6 but not by CCP2 and CCP3. Moreover, using 3EG antibody, we identify a C‑terminally truncated β-tubulin form with the same –EEEG C-terminal sequence. Using mass spectrometry, we demonstrate that β2A/B-tubulin is modified by truncation of the four C-terminal residues (EDEA). We show that this newly identified βΔ4-tubulin is ubiquitously present in cells and tissues and that its level is constant throughout the cell cycle. These new C-terminally truncated α- and β-tubulin variants, both ending with –EEEG sequence, are expected to regulate microtubule physiology. Of interest, the αΔ3-tubulin seems to be related to dynamic microtubules, resembling tyrosinated-tubulin rather than the other truncated variants, and may have critical function(s) in neuronal development.     

Expression pattern of estrogen receptors α and β and G-protein-coupled estrogen receptor 1 in the human testis

Estrogen signaling is considered to play an important role in spermatogenesis, spermiogenesis and male fertility. Estrogens can act via the two nuclear estrogen receptors ESR1 (ERα) and ESR2 (ERβ) or via the intracellular G-protein-coupled estrogen receptor 1 (GPER, formerly GPR30). Several reports on the localization and expression of all three receptors in the human testis have been published but are controversial particularly in case of ERα. Contrary to previous studies, we decided therefore to evaluate expression of all three receptors in the testis by a number of different methods and in comparison with MCF-7 cells. Using qPCR, we could show that mRNA expression of ERα is considerably lower and expression of ERβ and GPER much higher in the testis than in MCF-7 cells. RT-PCR after laser-assisted microdissection of tubular and interstitial compartments from normal and Sertoli cell only syndrome testes plus in situ hybridization and immunohistochemical analyses of the same samples demonstrated that there is very low expression of ERα in germ cells and in single interstitial cells, very high expression of ERβ in germ cells and Sertoli cells and high expression of GPER in interstitial cells and less in Sertoli cells.     

Identification and localization of estrogen receptor α- and β-positive cells in adult male and female mouse intestine at various estrogen levels

Although estrogen is implicated in the regulation of mammalian intestinal function, the presence and the distribution of estrogen receptor (ER)-positive cells in the intestine are still controversial. The present study was designed to localize ER- and ER-expressing cells in female and male mouse intestines immunohistochemically under various estrogen conditions, especially in female mice, ovariectomized as well at various phases of the estrous cycle. Western blot analysis detected both ER (66-kDa band) and ER (56-kDa band). Immunohistochemical staining of paraffin-embedded sections after antigen-retrieval treatment with autoclaving revealed staining for ER in submucosal interstitial cells, and double staining identified these cells as a subtype of intestinal macrophages. The number of these cells varied according to the estrous cycle phase. Administration of 17-estradiol to ovariectomized mice resulted in a significant increase in the number of ER-positive macrophages. On the other hand, the nuclei of nerve cells in Auerbach and Meissner plexuses were positive for both ER and ER, but the number of positive nerve cells was not affected by estrogen. Our results indicate that estrogen and estrogenic compounds may exert their actions on the intestine in two ways; one is through interstitial macrophages and the other is through intestinal neurons.     

Epigenetic setting for long-term expression of estrogen receptor α and androgen receptor in cells

Epigenetic regulation of the nuclear estrogen and androgen receptors, ER and AR, constitutes the molecular basis for the long-lasting effects of sex steroids on gene expression in cells. The effects prevail at hundreds of gene loci in the proximity of estrogen- and androgen-responsive elements and many more such loci through intra- and even inter-chromosomal level regulation. Such a memory system should be active in a flexible manner during the early development of vertebrates, and later replaced to establish more stable marks on genomic DNA. In mammals, DNA methylation is utilized as a very stable mark for silencing of the ERα and AR isoform expression during cancer cell and normal brain development. The factors affecting the DNA methylation of the ERα and AR genes in cells include estrogen and androgen. Since testosterone induces brain masculinization through its aromatization to estradiol in a narrow time window of the perinatal stage in rodents, the autoregulation of estrogen receptors, especially the predominant form of ERα, at the level of DNA methylation to set up the “cell memory” affecting the sexually differentiated status of brain function has been attracting increasing attention. The alternative usage of the androgen-AR system for brain masculinization and estrogenic regulation of AR expression in some species imply that the DNA methylation pattern of the AR gene can be established by closely related but different systems for sex steroid-induced phenomena, including brain masculinization.     

Localization of estrogen receptor α and progesterone receptor B in bovine cervix and vagina during the follicular and luteal phases of the sexual cycle

Abstract

The localization and distribution of estrogen receptors (ERα) and progesterone receptors (PR-B) in the cervix and vagina of sexually mature bovines during the follicular and luteal phases of the sexual cycle were studied using immunohistocehmistry. The estrous cycle stage of 23 Holstein bovines was assessed by gross and histological appearance of ovaries and blood steroid hormone values. Tissue samples from cervix and vagina were fixed in 10% formaldehyde for routine histological processing. Nuclear staining for ERα and PR-B was observed in the epithelial cells of the surface epithelium, stromal cells and smooth muscle cells. Generally, in the cervix, ERα immunoreactivity was more intense in the epithelial and smooth muscle cells during the follicular phase and in the epithelial cells during the luteal phase (p < 0.05). PR-B immunoreactivity was more intense in the epithelial and smooth muscle cells than in the superficial and deep stromal cells during the follicular and luteal phases (p < 0.05). In the vagina, ERα and PR-B immunoreactivities were more intense in the epithelial cells than in the connective tissue cells and smooth muscle cells during the follicular and luteal phases (p < 0.05). These results indicated that the frequency and intensity of ERα and PR-B immunoreactivity in the cervix and vagina of bovines varied according to the cervical and vaginal cell types and the phases of the sexual cycle.     

Post-hatching syrinx development in the zebra finch: an analysis of androgen receptor,aromatase, estrogen receptor α and estrogen receptor β mRNAs

In zebra finches, the vocal organ (syrinx) is larger in males than in females. Specific details about the mechanisms responsible for this dimorphism are not known, but may involve sex differences in steroid hormone action early in post-hatching development. The distribution of androgen receptor (AR), aromatase (AROM), estrogen receptor (ER), and estrogen receptor (ER) mRNAs was examined at post-hatching days 3, 10 and 17. A low level of AR was equivalently expressed in the syrinx muscles of both sexes at all three ages. We detected no specific expression of AROM or ER mRNAs. In contrast, ER mRNA was detected in chondrocytes of the forming bone. The density of this expression increased with age as the chondrocytes hypertrophied, but did not differ between the sexes. Taken together, these data suggest that estrogens may act on cartilage/bone, and androgens may act on muscle fibers in early post-hatching development to influence syrinx morphology. However, the lack of a sex difference in steroid receptor mRNA expression in the syrinx suggests that, similar to the forebrain regions that control song, the interaction of androgens and estrogens with their receptors is not sufficient to induce full sexual differentiation of this organ.     

α-Thalassemia and the production of different α chain variants in heterozygotes

The production of five chain variants (Hb G-Georgia, Hb St. Luke's, Hb Lloyd, Hb Montgomery, and Hb G-Philadelphia) in heterozygotes was evaluated through hematological observations, hemoglobin quantification, and biosynthetic studies. All heterozygotes for Hb St. Luke's and Hb Lloyd and most heterozygotes with Hb G-Georgia and Hb Montgomery had normal hematology and average / values of about 1.1. They were assigned a normal genotype (^G/), although the proportions of Hb St. Luke's and Hb G-Georgia were low (10 to 13%) and those of Hb Lloyd and Hb Montgomery twice as high (20%). Data from short-term incubations confirmed this genotype for some of these heterozygotes. Isolated Hb St. Luke's and Hb G-Georgia gave low ^G/ values (0.2 and 0.3) indicating that these Hb variants were defective at the level of Hb assembly. Isolated Hb Montgomery and Hb G-Philadelphia, however, gave higher ^G/ values of 0.6 and 0.8, respectively. A second type of variability existed among Hb G-Georgia (20 vs. 13%), Hb Montgomery (28 vs. 20%), and Hb G-Philadelphia (47 vs. 34%) heterozygotes, in whom the levels of Hb G differed. The occurrence of higher levels of these three chain heterozygosities was associated with hematological or biosynthetic evidence of a mild or moderate chain deficiency due to an -thalassemia-2 heterozygosity (^G/⁰ or ^0G/) or a homozygosity (^0G/⁰), respectively.This study was supported in part by USPHS Research Grants HLB-05168 and HLB-15158.     

Gender- and region-specific variations of estrogen receptor α and β expression in the growth plate of spine and limb during development and adulthood

Although estrogen action is indispensable for normal bone growth in both genders, the roles of estrogen receptors (ERs) in mediating bone growth are not fully understood. The effects of ER inactivation on bone growth are sex and age dependent, and may differ between the axial and appendicular regions. In this study, the spatial and temporal expression of ERα and β in the tibial and spinal growth plates of the female and male rats during postnatal development was examined to explore the possible mechanisms. The level of mRNA was examined and compared with quantitative real-time PCR. The spatial location was determined by immunohistochemical analysis. The 1-, 4-, 7-, 12- and 16-week age stages correspond to early life, puberty and early adulthood after puberty, respectively. Gender- and region-specific differences in ERα and β expression were shown in the growth plates. Mainly nuclear staining of ERα and β immunoreactivity was demonstrated in the spinal and tibial growth plate chondrocytes for both genders. Moreover, our study indicated significant effect of gender on temporal ERα and β expression and of region on temporal ERα/ERβ expression ratio. However, spatial differences of region-related ERα and β expression were not observed. Gender-related spatial changes were detected only at 16 weeks of both spine and limb growth plates. ERα and β immunoreactivity was detected in the resting, proliferative and prehypertrophic chondrocytes in the early life stage and during puberty. After puberty, ERα expression was mainly located in the late proliferative and hypertrophic chondrocytes in female, whereas the expression still extended from the resting to hypertrophic chondrocytes in males. Gender- and region-specific expression patterns of ERα and β gene might be one possible reason for differences in sex- and region-related body growth phenotypes. Gender, age and region differences should be taken into consideration when the roles of ERs in the growth plate are investigated.     

Expression of extracellular matrix components is disrupted in the immature and adult estrogen receptor β-null mouse ovary

Within the ovary, Estrogen Receptor β (ERβ) is localized to the granulosa cells of growing follicles. 17β-estradiol (E2) acting via ERβ augments the actions of follicle stimulating hormone in granulosa cells, leading to granulosa cell differentiation and formation of a preovulatory follicle. Adult ERβ-null females are subfertile and possess ovaries with reduced numbers of growing follicles and corpora lutea. Because the majority of E2 production by granulosa cells occurs once puberty is reached, a role for ERβ in the ovary prior to puberty has not been well examined. We now provide evidence that lack of ERβ disrupts gene expression as early as post-natal day (PND) 13, and in particular, we identify a number of genes of the extracellular matrix (ECM) that are significantly higher in ERβ-null follicles than in wildtype (WT) follicles. Considerable changes occur to the ECM occur during normal folliculogenesis to allow for the dramatic growth, cellular differentiation, and reorganization of the follicle from the primary to preovulatory stage. Using quantitative PCR and immunofluorescence, we now show that several ECM genes are aberrantly overexpressed in ERβ-null follicles. We find that Collagen11a1, a protein highly expressed in cartilage, is significantly higher in ERβ-null follicles than WT follicles as early as PND 13, and this heightened expression continues through PND 23-29 into adulthood. Similarly, Nidogen 2, a highly conserved basement membrane glycoprotein, is elevated in ERβ-null follicles at PND 13 into adulthood, and is elevated specifically in the ERβ-null focimatrix, a basal lamina-like matrix located between granulosa cells. Focimatrix laminin and Collagen IV expression were also higher in ERβ-null ovaries than in WT ovaries at various ages. Our findings suggest two novel observations: a) that ERβ regulates granulosa cell gene expression ovary prior to puberty, and b) that ERβ regulates expression of ECM components in the mouse ovary.     

Identification and characterization of lipopolysaccharide binding protein (LBP) as an estrogen receptor α specific serum biomarker

Estrogen Receptor α (ERα) and Estrogen Receptor β (ERβ) are steroid nuclear receptors that transduce estrogen signaling to control diverse physiological processes linked to reproduction, bone remodeling, behavior, immune response and endocrine-related diseases. In order to differentiate between ERα and ERβ mediated effects in vivo, ER subtype selective biomarkers are essential. We utilized ERα knockout (AERKO) and ERβ knockout (BERKO) mouse liver RNA and genome wide profiling to identify novel ERα selective serum biomarker candidates. Results from the gene array experiments were validated using real-time RT-PCR and subsequent ELISA's to demonstrate changes in serum proteins. Here we present data that Lipopolysacharide Binding Protein (LBP) is a novel liver-derived ERα selective biomarker that can be measured in serum.     

Expression of estrogen receptor α and β in rat astrocytes in primary culture: effects of hypoxia and glucose deprivation

Estrogen replacement therapy could play a role in the reduction of injury associated with cerebral ischemia in vivo, which could be, at least partially, a consequence of estrogen influence of glutamate buffering by astrocytes during hypoxia/ischemia. Estrogen exerts biological effects through interaction with its two receptors: estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ), which are both expressed in astrocytes. This study explored effects of hypoxia and glucose deprivation (HGD), alone or followed by 1 h recovery, on ERα and ERβ expression in primary rat astrocyte cultures following 1 h exposure to: a) 5 % CO(2) in air (control group-CG); b) 2 % O(2)/5 % CO(2) in N(2) with glucose deprivation (HGD group-HGDG); or c) the HGDG protocol followed by 1 h CG protocol (recovery group-RG). ERα mRNA expression decreased in HGDG. At the protein level, full-length ERα (67 kDa) and three ERα-immunoreactive protein bands (63, 60 and 52 kDa) were detected. A significant decrease in the 52 kDa band was seen in HGDG, while a significant decrease in expression of the full length ERα was seen in the RG. ERβ mRNA and protein expression (a 54 kDa single band) did not change. The observed decrease in ERα protein may limit estrogen-mediated signalling in astrocytes during hypoxia and recovery.