17β-estradiol modulates NGF and BDNF expression through ERβ mediated ERK signaling in cortical astrocytes

17β-estradiol is known to exert neurotrophic and neuroprotective effects through classical estrogen receptors [ERs], ERα and ERβ, on a variety of cell types either by genomic or non-genomic actions. The actions of estradiol on glial cells are important to maintain metabolic functions of the nervous system. Astrocytes are considered to be active participants in brain activity because of their ability to release growth factors, including neurotrophins. Present in vitro studies show that 17β-estradiol modulates NGF and BDNF expression in time-dependent manner and ERK acts as secondary messenger for estradiol’s action. 17β-estradiol is involved in survival of cortical astrocytes. In conclusion, this study indicates vital role of ERβ mediated ERK signalling for regulation of NGF and BDNF expression along with cell viability of cortical astrocytes which further confirms the role of ERs, particularly ERβ in glial cells’ functions and viability.     

3D models of human ERα and ERβ complexed with 5-androsten-3β,17β-diol

Recently, binding of 5-androsten-3β,17β-diol (Δ(5)-androstenediol) to human estrogen receptor-beta (ERβ) was found to repress microglia-mediated inflammation, which is associated with various neurodegenerative diseases, such as multiple sclerosis. In contrast, binding of estradiol to ERβ resulted in little or no repression of microglia-mediated inflammation. Binding of Δ(5)-androstenediol to ERβ, as well as to ERα, is unexpected because unlike estradiol, Δ(5)-androstenediol has a saturated A ring and a C19 methyl group. To begin to elucidate the interaction of Δ(5)-androstenediol with both ERs, we constructed 3D models of Δ(5)-androstenediol with human ERα and ERβ for comparison with the crystal structures of estradiol in ERα and ERβ. Conformational flexibility in human ERα and ERβ accommodates the C19 methyl on Δ(5)-androstenediol. This conformational flexibility may be relevant for binding of other Δ(5)-steroids with C19 methyl substituents, such as 25-hydroxycholesterol and 27-hydroxycholesterol, to ERs.     

ERβ protein expression in female cynomolgus monkey and CF‐1 mouse brain: Western analysis

In humans and rodents, multiple ERβ variants with sizes ranging from 477–549 amino acids (aa) have been described. The identification of these variants in target tissues has important implications for estrogen signaling and cellular responsiveness. Western blot analysis using two anti‐ERβ antibodies specific for mammalian ERβ sequences (PA1‐310B and PA1‐311) was employed to examine ERβ protein expression in neural tissues from ovariectomized (OVX) cynomolgus macaques and CF‐1 mice as well as to assess potential regulatory effects of acute and extended estradiol (E₂) treatment. In hypothalamic extracts from both species, a single ERβ immunoreactive (ERβ‐ir) band was detected at approximately 54 kDa, corresponding to the expected molecular weight for ERβ477 and/or 485. In cynomolgus females, oral E₂ administration for 16 weeks had no apparent effect on hypothalamic ERβ protein expression. In mouse, a single injection of E₂ did not change hypothalamic ERβ protein levels 1.5, 4, 8, 16, or 24 h after injection. Extending the hormonal treatment to 4 or 21 days in OVX female mice also had no effect on the level of hypothalamic ERβ protein. Additional regional analyses in female mouse brain with PA1‐310B antibody showed that a second, 59 kDa ERβ‐ir band was present in cortex, striatum, hippocampus, and amygdala that could represent one or both of the larger ERβ variants (530 and 549aa). The expression level of the second ERβ isoform exhibited regional variation, with the strongest immunoreactivity detected in cortex and amygdala. Elucidating the functions of these ERβ isoforms in the CNS will facilitate our understanding of the tissue‐ and promoter‐specific actions of estrogen. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2005     

Estrogen‐regulated developmental neuronal apoptosis is determined by estrogen receptor subtype and the Fas/Fas ligand system

Adult sexual dimorphism in neuronal cell number is controlled by estrogen exposure during a tightly defined period of rat brain development. The mechanisms of estrogen's effect are unknown; one possibility is regulation of programmed cell death (apoptosis). In this study we have shown that estradiol can function as a neuroprotective agent or an inducer of apoptosis, depending on the estrogen receptor‐subtype present in the cell. Thus, ERα has a neuroprotective effect, while ERβ mediates the induction of apoptosis in neuronal cells. Moreover, we show that estrogen‐induced apoptosis through ER‐β requires the expression of Fas‐ and Fas ligand (FasL) proteins, since the absence of FasL in neurons prevents this effect. Furthermore, we demonstrate that microglia‐secreted products induce the expression of FasL necessary to mediate estradiol–ERβ apoptotic effect. These findings may explain the dichotomous effect of fetal estradiol on the adult neuronal number. © 2000 John Wiley & Sons, Inc. J Neurobiol 43: 64–78, 2000     

Synthesis and evaluation of aryl-substituted diarylpropionitriles,selective ligands for estrogen receptor β, as positron-emission tomographic imaging agents

We have investigated halogen-substituted non-steroidal estrogens with selective binding affinity for the estrogen receptor β (ERβ that might be used for imaging the levels of this ER-subtype in breast tumors by positron emission tomography (PET). Based on diarylpropionitrile (DPN, 1a), a compound previously reported that has a 72-fold binding selectivity for ERβ, we developed a series of DPN analogs having methyl-, hydroxyl-, and halogen substituents, including fluoroethyl and fluoropropyl groups. In competitive radiometric binding assays with [³H]estradiol, all of these DPN analogs showed high ERβ/ERα selectivity; while the selectivity varied, in some cases it reached nearly 300-fold (RBA: ERα, 0.023%; ERβ, 6.25%). The absolute ERβ binding affinities, however, were not sufficient to merit further consideration for developing these ligands as PET imaging agents.     

Synthesis and evaluation of 17α-triazolyl and 9α-cyano derivatives of estradiol

A variety of 17α-triazolyl and 9α-cyano derivatives of estradiol were prepared and evaluated for binding to human ERβ in both a TR-FRET assay, as well as ERβ and ERα agonism in cell-based functional assays. 9α-Cyanoestradiol (5) was nearly equipotent as estradiol as an agonist for both ERβ and ERα. The potency of the 17α-triazolylestradiol analogs is considerably more variable and depends on the nature of the 4-substituent of the triazole ring. While rigid protein docking simulations exhibited significant steric clashing, induced fit docking providing more protein flexibility revealed that the triazole linker of analogs 2d and 2e extends outside of the traditional ligand binding domain with the benzene ring located in the loop connecting helix 11 to helix 12.     

Sites of in vivo extraction and interconversion of estrone and estradiol in the dog

The interconversion and extraction of estrone and estradiol-17β across and within different tissues or areas have been studied in the dog by the constant infusion technique. The results were calculated using the ³H/¹⁴C ratios and radioactive concentrations of estrone and estradiol obtained from afferent and efferent blood and tissues at equilibrium. From these results it is concluded that: (1) there is no significant difference between metabolic clearance rates of estrone and estradiol, (2) blood transfer constants indicate a higher conversion of estradiol to estrone than of estrone to estradiol, (3) the transtissue interconversion favors the formation of estrone while the intratissue interconversion favors the formation of estradiol, (4) no interconversion of the two estrogens is observed in adipose tissue, (5) the extraction of estradiol entering a tissue was lower than the extraction of estradiol formed in these tissues, (6) calculation of the tissue metabolic clearance rates show that 63% and 61% of the total metabolism of estrone and estradiol, respectively, occurs in the splanchnic bed, and (7) the contribution of each tissue to the total interconversion of estrone and estradiol show that more than 90% of this interconversion occurs extrahepatically.     

Sex-associated difference in estrogen receptor β expression in N-methyl-N'-nitro-N-nitrosoguanidine-induced gastric cancers in rats

Epidemiologic studies indicate that the incidence of gastric cancer is higher in males than in females. Although the mechanisms mediating this difference are unclear, a role for estrogens has been proposed. We used Western blotting to evaluate the role of estrogen receptor (ER) subtypes ERα and ERβ and proliferating cell nuclear antigen (PCNA) in N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced gastric carcinogenesis in Wistar rats; ERα and ERβ mRNA levels also were analyzed by quantitative real-time RT-PCR analysis. The incidence of gastric cancer was significantly higher in male than female rats. In both sexes, ERα expression was similar in MNNG-treated cancerous and noncancerous tissues and normal gastric tissue. However, ERβ expression in MNNG-treated cancerous and noncancerous tissues was significantly lower in male rats and higher in female rats than that in normal gastric tissue; MNNG-induced cancerous tissue showed the highest ERβ expression. PCNA expression in MNNG-treated cancerous tissues was higher than that in noncancerous tissues, and was higher in male rats than female rats. Western blotting results were consistent with the mRNA changes determined by quantitative real-time RT-PCR. The present study provides evidence of a sex-associated difference in ERβ and PCNA expression in MNNG-induced gastric cancers in Wistar rats.     

Influence of ERβ selective agonism during the neonatal period on the sexual differentiation of the rat hypothalamic-pituitary-gonadal (HPG) axis

Background

It is well established that sexual differentiation of the rodent hypothalamic-pituitary-gonadal (HPG) axis is principally orchestrated by estrogen during the perinatal period. Here we sought to better characterize the mechanistic role the beta form of the estrogen receptor (ERβ) plays in this process.

Methods

To achieve this, we exposed neonatal female rats to three doses (0.5, 1 and 2 mg/kg) of the ERβ selective agonist diarylpropionitrile (DPN) using estradiol benzoate (EB) as a positive control. Measures included day of vaginal opening, estrous cycle quality, GnRH and Fos co-localization following ovariectomy and hormone priming, circulating luteinizing hormone (LH) levels and quantification of hypothalamic kisspeptin immunoreactivity. A second set of females was then neonatally exposed to DPN, the ERα agonist propyl-pyrazole-triol (PPT), DPN+PPT, or EB to compare the impact of ERα and ERβ selective agonism on kisspeptin gene expression in pre- and post-pubescent females.

Results

All three DPN doses significantly advanced the day of vaginal opening and induced premature anestrus. GnRH and Fos co-labeling, a marker of GnRH activation, following ovariectomy and hormone priming was reduced by approximately half at all doses; the magnitude of which was not as large as with EB or what we have previously observed with the ERα agonist PPT. LH levels were also correspondingly lower, compared to control females. No impact of DPN was observed on the density of kisspeptin immunoreactive (-ir) fibers or cell bodies in the arcuate (ARC) nucleus, and kisspeptin-ir was only significantly reduced by the middle (1 mg/kg) DPN dose in the preoptic region. The second experiment revealed that EB, PPT and the combination of DPN+PPT significantly abrogated preoptic Kiss1 expression at both ages but ARC expression was only reduced by EB.

Conclusion

Our results indicate that selective agonism of ERβ is not sufficient to completely achieve male-typical HPG organization observed with EB or an ERα agonist.     

Estrogen negatively regulates epithelial wound healing and protective lipid mediator circuits in the cornea

Estrogen receptors (ERs) are expressed in leukocytes and in every ocular tissue. However, sex-specific differences and the role of estradiol in ocular inflammatory-reparative responses are not well understood. We found that female mice exhibited delayed corneal epithelial wound closure and attenuated polymorphonuclear (PMN) leukocyte responses, a phenotype recapitulated by estradiol treatment both in vivo (topically in male mice) and in vitro (corneal epithelial cell wound healing). The cornea expresses 15-lipoxygenase (15-LOX) and receptors for lipoxin A(4) (LXA(4)), which have been implicated in an intrinsic lipid circuit that regulates corneal inflammation and wound healing. Delayed epithelial wound healing correlated with lower expression of 15-LOX in the regenerated epithelium of female mice. Estradiol in vitro and in vivo down-regulated epithelial 15-LOX expression and LXA(4) formation, while estradiol abrogation of epithelial wound healing was completely reversed by treatment with LXA(4). More important, ERβ and ERα selectively regulated epithelial wound healing, PMN cell recruitment, and activity of the intrinsic 15-LOX/LXA(4) circuit. Our results demonstrate for the first time a sex-specific difference in the corneal reparative response, which is mediated by ERβ and ERα selective regulation of the epithelial and PMN 15-LOX/LXA(4) circuit. These findings may provide novel insights into the etiology of sex-specific ocular inflammatory diseases.     

Opposite effects of two estrogen receptors on tau phosphorylation through disparate effects on the miR‐218/PTPA pathway

The two estrogen receptors (ERs), ERα and ERβ, mediate the diverse biological functions of estradiol. Opposite effects of ERα and ERβ have been found in estrogen‐induced cancer cell proliferation and differentiation as well as in memory‐related tasks. However, whether these opposite effects are implicated in the pathogenesis of Alzheimer's disease (AD) remains unclear. Here, we find that ERα and ERβ play contrasting roles in regulating tau phosphorylation, which is a pathological hallmark of AD. ERα increases the expression of miR‐218 to suppress the protein levels of its specific target, protein tyrosine phosphatase α (PTPα). The downregulation of PTPα results in the abnormal tyrosine hyperphosphorylation of glycogen synthase kinase‐3β (resulting in activation) and protein phosphatase 2A (resulting in inactivation), the major tau kinase and phosphatase. Suppressing the increased expression of miR‐218 inhibits the ERα‐induced tau hyperphosphorylation as well as the PTPα decline. In contrast, ERβ inhibits tau phosphorylation by limiting miR‐218 levels and restoring the miR‐218 levels antagonized the attenuation of tau phosphorylation by ERβ. These data reveal for the first time opposing roles for ERα and ERβ in AD pathogenesis and suggest potential therapeutic targets for AD.     

Characterization of metabolic health in mouse models of fibrillin-1 perturbation

Mutations in the microfibrillar protein fibrillin-1 or the absence of its binding partner microfibril-associated glycoprotein (MAGP1) lead to increased TGFβ signaling due to an inability to sequester latent or active forms of TGFβ, respectively. Mouse models of excess TGFβ signaling display increased adiposity and predisposition to type-2 diabetes. It is therefore interesting that individuals with Marfan syndrome, a disease in which fibrillin-1 mutation leads to aberrant TGFβ signaling, typically present with extreme fat hypoplasia. The goal of this project was to characterize multiple fibrillin-1 mutant mouse strains to understand how fibrillin-1 contributes to metabolic health. The results of this study demonstrate that fibrillin-1 contributes little to lipid storage and metabolic homeostasis, which is in contrast to the obesity and metabolic changes associated with MAGP1 deficiency. MAGP1 but not fibrillin-1 mutant mice had elevated TGFβ signaling in their adipose tissue, which is consistent with the difference in obesity phenotypes. However, fibrillin-1 mutant strains and MAGP1-deficient mice all exhibit increased bone length and reduced bone mineralization which are characteristic of Marfan syndrome. Our findings suggest that Marfan-associated adipocyte hypoplasia is likely not due to microfibril-associated changes in adipose tissue, and provide evidence that MAGP1 may function independently of fibrillin in some tissues.     

Proliferation assays for estrogenicity testing with high predictive value for the in vivo uterotrophic effect

Proliferation assays based on human cell lines are the most used in vitro tests to determine estrogenic properties of compounds. Our objective was to characterise to what extent these in vitro tests provide alternatives for the in vivo Allen and Doisy test, a uterotrophic assay in immature or ovariectomised rodents with uterus weight as a crucial read-out parameter. In the present study four different human cell lines derived from three different female estrogen-sensitive tissues, i.e. breast (MCF-7/BOS and T47D), endometrial (ECC-1) and ovarian (BG-1) cells, were characterised by investigating their relative ERα and ERβ amounts, as the ERα/ERβ ratio is a dominant factor determining their estrogen-dependent proliferative responses. All four cell lines clearly expressed the ERα type and a very low but detectable amount of ERβ on both the mRNA and protein level, with the T47D cell line expressing the highest level of the ERβ type. Subsequently, a set of reference compounds representing different modes of estrogen action and estrogenic potency were used to investigate the proliferative response in the four cell lines, to determine which cell line most accurately predicts the effect observed in vivo. All four cell lines revealed a reasonable to good correlation with the in vivo uterotrophic effect, with the correlation being highest for the MCF-7/BOS cell line (R2=0.85). The main differences between the in vivo uterotrophic assay and the in vitro proliferation assays were observed for tamoxifen and testosterone. The proliferative response of the MCF-7/BOS cells to testosterone was partially caused by its conversion to estradiol by aromatase or via androstenedione to estrone. It is concluded that of the four cell lines tested, the best assay to include in an integrated testing strategy for replacement of the in vivo uterotrophic assay is the human MCF-7/BOS breast cancer cell line.     

In vitro and in vivo molecular imaging of estrogen receptor α and β homo- and heterodimerization: exploration of new modes of receptor regulation

Estrogen receptor (ER) biology reflects the actions of estrogens through the two receptors, ERα and ERβ, although little is known regarding the preference for formation of ER homo- vs. heterodimers, and how this is affected by the level of ligand occupancy and preferential ligand affinity for one of the ER subtypes. In this report, we use a split optical reporter-protein complementation system to demonstrate the physical interaction between ERα and ERβ in response to different ER ligands in cells and, for the first time, by in vivo imaging in living animals. The genetically encoded reporter vectors constructed with the ligand-binding domains of ERα and ERβ, fused to split firefly or Renilla luciferase (Fluc or hRluc) fragments, were used for this study. This molecular proteomic technique was used to detect ERα/ERα or ERβ/ERβ homodimerization, or ERα/ERβ heterodimerization induced by ER subtype-selective and nonselective ligands, and selective ER modulators (SERM), as well as in dimers in which one mutant monomer was unable to bind estradiol. The SERM-bound ERα and ERβ form the strongest dimers, and subtype-preferential homodimerization was seen with ERα-selective ligands (methyl piperidino pyrazole/propyl pyrazole triol) and the ERβ-selective ligands (diarylpropionitrile/tetrahydrochrysene/genistein). We also demonstrated that a single ligand-bound monomer can form homo- or heterodimers with an apo-monomer. Xenografts of human embryonic kidney 293T cells imaged in living mice by bioluminescence showed real-time ligand induction of ERα/ERβ heterodimerization and reversal of dimerization upon ligand withdrawal. The results from this study demonstrate the value of the split luciferase-based complementation system for studying ER-subtype interactions in cells and for evaluating them in living animals by noninvasive imaging. They also probe what combinations of ERα and ERβ dimers might be the mediators of the effects of different types of ER ligands given at different doses.