Rapid action of estradiol in primate GnRH neurons: the role of estrogen receptor alpha and estrogen receptor beta

Estrogens play a pivotal role in the control of female reproductive function. Recent studies using primate GnRH neurons derived from embryonic nasal placode indicate that 17β-estradiol (E₂) causes a rapid stimulatory action. E₂ (1 nM) stimulates firing activity and intracellular calcium ([Ca²⁺]_i) oscillations of primate GnRH neurons within a few min. E₂ also stimulates GnRH release within 10 min. However, the classical estrogen receptors, ERα and ERβ, do not appear to play a role in E₂-induced [Ca²⁺]_i oscillations or GnRH release, as the estrogen receptor antagonist, ICI 182,780, failed to block these responses. Rather, this rapid E₂ action is, at least in part, mediated by a G-protein coupled receptor GPR30. In the present study we further investigate the role of ERα and ERβ in the rapid action of E₂ by knocking down cellular ERα and ERβ by transfection of GnRH neurons with specific siRNA for rhesus monkey ERα and ERβ. Results indicate that cellular knockdown of ERα and ERβ failed to block the E₂-induced changes in [Ca²⁺]_i oscillations. It is concluded that neither ERα nor ERβ is required for the rapid action of E₂ in primate GnRH neurons.     

Involvement of IGF-I receptor and estrogen receptor pathways in the protective effects of ginsenoside Rg1 against Aβ25–35-induced toxicity in PC12 cells

Ginsenoside Rg1 is the main pharmacologically active compound of ginsenosides and has demonstrated pharmacological effects in the cardiovascular system, central nervous system and immune system. The involvement of insulin-like growth factor-I receptor (IGF-IR)-dependent pathway and estrogen receptor (ER)-dependent pathway in the biological effect of ginsenoside Rg1 have been demonstrated in our previous study. The present study tested the hypothesis that the protective effects of Rg1 against Aβ_25–35-induced toxicity involved activation of the IGF-IR and ER signaling pathways in PC12 cells. Treatment with Aβ_25–35 decreased the cell viability in a dose-dependent manner in PC12 cells. Rg1 pretreatment resulted in an enhancement of survival and the maximum protection occurred at the concentration of 1 μM. Co-treatment with IGF-IR antagonist JB-1 or ER antagonist ICI182,780 could completely block the protective effect of Rg1. The decreased Bcl-2 mRNA expression induced by Aβ_25–35 could be restored by Rg1 pretreatment. Rg1 pretreatment could also restore the decreased mitochondrial membrane potential induced by Aβ_25–35 and these effects could be completely blocked by JB-1 or ICI182,780. In addition, Rg1 treatment alone could significantly increase the phosphorylation level of MEK and ERK in a time-dependent manner and the functional transactivation of ERα in PC12 cells. The functional transactivation of ERα by Rg1 could be completely blocked by JB-1 or ICI182,780. Taken together, our results suggest that IGF-IR and ER signaling pathways might be involved in the protective effect of Rg1 against Aβ_25–35-induced toxicity in PC12 cells.     

Estrogenic activities of Psoralea corylifolia L. seed extracts and main constituents

Estrogenic activities of ethanol extract and its active components from Psoralea corylifolia L. were studied using various in vitro assays. The main components from ethanol extract were analyzed to be bakuchiol, psoralen, isobavachalcone, isobavachromene, and bavachinin. In a fractionation procedure, hexane and chloroform fractions showed estrogenic activity in yeast transactivation assay and E-screen assay. In yeast transactivation assay, ethanol extract, hexane, and chloroform fractions showed significantly higher activities at a concentration of 1.0 ng/ml, and bakuchiol at the concentration of 10⁻⁶ M was showed the highest activity, especially, which was higher than genistein at the same concentration. In E-screen assay, cell proliferation of bakuchiol (10⁻⁶ M) showed similar estrogenic activity with genistein (10⁻⁶ M). In ER binding assay, bakuchiol displayed the strongest ER-binding affinity (IC₅₀ for ERα = 1.01 × 10⁻⁶ M, IC₅₀ for ERβ = 1.20 × 10⁻⁶ M) and bakuchiol showed five times higher affinity for ERα than for ERβ.     

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin, 12-O-tetradecanoylphorbol-13-acetate and 17β-estradiol on estrogen receptor regulation in MCF-7 human breast cancer cells

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) exhibits remarkably potent antiestrogenic activity. To further elucidate the role of estrogen receptor (ER) regulation in this response, we examined the effects of exposure to TCDD in MCF-7 human breast cancer cells on ER mRNA levels by using an RNase protection assay, on ER accumulation by using an ER immunocytochemical essay (ER-ICA), and on ER function by competitive binding assays under conditions of saturating 17β-estradiol (E₂). Comparative studies were conducted with E₂ and 12-O-tetradecanoylphorbol-13-acetate (TPA), as both compounds are known to suppress ER expression. Our results indicate that 1 nM E₂ and 100 nM TPA both suppress ER mRNA levels as early as 4 h after exposure and to 33.6% and 16.5% of control levels, respectively, after 72 h. In contrast, no significant effect on ER mRNA levels was attributed to exposure to 10 nM TCDD. A greater than 50% reduction in positive staining was observed by ER-ICA after 72 h exposure to 1 nM E₂ and to 100 nM TPA, while only an 11% reduction in positive staining was observed with 10 nM TCDD. Specific binding of [³H]E₂ under saturating conditions (10 nM E₂) in whole cells was reduced by 50% in cultures exposed to 100 nM TPA, although no effect on binding was observed with exposure to 10 nM TCDD. In contrast, specific binding using subsaturating 1 nM [³H]E₂ was depressed by 49% in MCF-7 cells exposed to 10 nM TCDD for 72 h. This depression was inhibited by a 1-h treatment with 5 μM α-naphthoflavone, which inhibits TCDD-induced, P450-mediated, E₂ metabolism, and subsequent E₂ depletion. In conclusion, while TPA and E₂ effectively down-regulate ER expression, TCDD, under antiestrogenic conditions, has little if any effect on total ER levels in MCF-7 cells, and thus ER modulation is probably not necessary for the suppression of estrogenic activity in MCF-7 cells by TCDD. © 1996 Wiley-Liss, Inc.     

A novel NADP-dependent dehydrogenase activity for 7α/β- and 11β-hydroxysteroids in human liver nuclei: A third 11β-hydroxysteroid dehydrogenase

Human tissue from uninvolved liver of cancer patients was fractionated using differential centrifugation and characterized for 11βHSD enzyme activity against corticosterone, dehydrocorticosterone, 7α- and 7β-hydroxy-dehydroepiandrosterone, and 7-oxo-dehydroepiandrosterone. An enzyme activity was observed in nuclear protein fractions that utilized either NADP⁺ or NAD⁺, but not NADPH and NADH, as pyridine nucleotide cofactor with K_m values of 12 ± 2 and 390 ± 2 μM, compared to the K_m for microsomal 11βHSD1 of 43 ± 8 and 264 ± 24 μM, respectively. The K_m for corticosterone in the NADP⁺-dependent nuclear oxidation reaction was 102 ± 16 nM, compared to 4.3 ± 0.8 μM for 11βHSD1. The K_cat values for nuclear activity with NADP⁺ was 1687 nmol/min/mg/μmol, compared to 755 nmol/min/mg/μmol for microsomal 11βHSD1 activity. Inhibitors of 11βHSD1 decreased both nuclear and microsomal enzyme activities, suggesting that the nuclear activity may be due to an enzyme similar to 11βHSD Type 1 and 2.     

Kinetics of the Ca, H, and Mg Interaction with the Ion-Binding Sites of the SR Ca-ATPase

Electrochromic styryl dyes were used to investigate mutually antagonistic effects of Ca²⁺ and H⁺ on binding of the other ion in the E₁ and P-E₂ states of the SR Ca-ATPase. On the cytoplasmic side of the protein in the absence of Mg²⁺ a strictly competitive binding sequence, H₂E₁?HE₁?E₁?CaE₁?Ca₂E₁, was found with two Ca²⁺ ions bound cooperatively. The apparent equilibrium dissociation constants were in the order of K_1/2(2 Ca) = 34 nM, K_1/2(H) = 1 nM and K_1/2(H₂) = 1.32 μM. Up to 2 Mg²⁺ ions were also able to enter the binding sites electrogenically and to compete with the transported substrate ions (K_1/2(Mg) = 165 μM, K_1/2(Mg₂) = 7.4 mM). In the P-E₂ state, with binding sites facing the lumen of the sarcoplasmatic reticulum, the measured concentration dependence of Ca²⁺ and H⁺ binding could be described satisfactorily only with a branched reaction scheme in which a mixed state, P-E₂CaH, exists. From numerical simulations, equilibrium dissociation constants could be determined for Ca²⁺ (0.4 mM and 25 mM) and H⁺ (2 μM and 10 μM). These simulations reproduced all observed antagonistic concentration dependences. The comparison of the dielectric ion binding in the E₁ and P-E₂ conformations indicates that the transition between both conformations is accompanied by a shift of their (dielectric) position.     

Activation of the human estrogen receptor by estrogenic and antiestrogenic compounds in Saccharomyces cerevisiae: a positive selection system

The yeast URA3 gene was used as a reporter to investigate the activities of estrogenic and antiestrogenic compounds in yeast Saccharomyces cerevisiae. The control sequences of the wild type (wt) URA3 promoter were replaced with zero, two, or six copies of estrogen-response elements (ERE). Insertion of two and six copies of ERE rendered the expression of the URA3 gene to be dependent on the presence of the human estrogen receptor (ER) and the hormone 17β-estradiol (E₂). Two versions of the ER genes were constructed: a full-length wild-type ER (ERa-f) and a truncated ER with domains C, D, and E (ERcde). Both forms of the ER were able to activate the ERE-URA3 reporter in a hormone-dependent manner. The growth of yeast transformants were hormone-dependent when the reporter constructs were inserted into chromosomes using yeast integrating vectors (YIp) but not with the 2μ-based episomal (high-copy number, YEp) or centromeric (low-copy number, YCp) vectors. The integrated transformants were employed to investigate the effects of estrogenic and antiestrogenic compounds. The estrogenic compounds, E₂, diethylstilbestrol (DES), and estrone (EST), activated expression of the reporter genes at 1 nM concentration, which is the same concentration exhibiting activity in mammalian cells. None of the antiestrogens, at concentrations up to 1 μM, including tamoxifen (TAM), raloxifene (RAL), and ICI 164,384 (ICI) antagonized 1 nM of E₂ against either form of the ER. In fact, TAM, RAL, and ICI displayed slight agonistic activity at high concentrations of 300 nM or greater to the ERcde. This system can be used to investigate or clone the missing factor(s) that is responsible for the antagonistic activity of the ER in yeast, and is also suitable for screening for the effectors of the ER.     

The thiol proteinase inhibitor E-64-d ameliorates amyloid-β-induced reduction of sAPPα secretion by reversing ceramide-induced protein kinase C down-regulation in SH-SY5Y neuroblastoma cells

In Alzheimer’s disease (AD), enhancing α-secretase processing of amyloid precursor protein (APP) is an important pathway to decrease neurotoxic amyloid β (Aβ) secretion. The α-secretase is reported to be regulated by protein kinase C (PKC) and various endogenous proteins or cell surface receptors. In this report, we first examined whether Aβ reduces α-secretase activity, and showed that Aβ peptide 1–40 (0.001 and 0.01 μM) reduced the secretion of soluble amyloid precursor protein α (sAPPα) in carbachol-stimulated SH-SY5Y neuroblastoma cells. E-64-d (3 μM), which is a potent calpain inhibitor that prevents PKC degradation, ameliorated the Aβ-induced reduction of sAPPα secretion. In addition, we observed that Aβ significantly enhanced ceramide production by activating neutral sphingomyelinase. The cell-permeable ceramide analog, C₂-ceramide (1 μg/mL), also reduced sAPPα secretion, and in addition, E-64-d eliminated the observed decrease of sAPPα secretion. C₂-ceramide induced down-regulation of PKC-α, -β₁, and -β₂ isozymes in SH-SY5Y cells. These findings suggest that ceramide may play an important role in sAPPα processing by modulating PKC activity.     

Role of 11β-OH-C(19) and C(21) steroids in the coupling of 11β-HSD1 and 17β-HSD3 in regulation of testosterone biosynthesis in rat Leydig cells

Here we describe further experiments to support our hypothesis that bidirectional 11β-HSD1-dehydrogenase in Leydig cells is a NADP(H) regenerating system. In the absence of androstenedione (AD), substrate for 17β-HSD3, incubation of Leydig cells with corticosterone (B) or several C₁₉- and C₂₁-11β-OH-steroids, in the presence of [³H]-11-dehydro-corticosterone (A), stimulated 11β-HSD1-reductase activity. However, in presence of 30 μM AD, testosterone (Teso) synthesis is stimulated from 4 to 197 picomole/25,000 cells/30 min and concomitantly inhibited 11β-HSD1-reductase activity, due to competition for the common cofactor NADPH needed for both reactions. Testo production was further significantly increased (p < 0.05) to 224-267 picomole/25,000 cells/30 min when 10 μM 11β-OH-steroids (in addition to 30 μM AD) were also included. Similar results were obtained in experiments conducted with lower concentrations of AD (5 μM), and B or A (500 nM).Incubations of 0.3-6.0 μM of corticosterone (plus or minus 30 μM AD) were then performed to test the effectiveness of 17β-HSD3 as a possible NADP⁺ regenerating system. In the absence of AD, increasing amounts (3-44 pmol/25,000 cells/30 min) of 11-dehydro-corticosterone were produced with increasing concentrations of corticosterone in the medium. When 30 μM AD was included, the rate of 11-dehydro-corticosterone formation dramatically increased 1.3-5-fold producing 4-210 pmol/25,000 cells/30 min of 11-dehydro-corticosterone. We conclude that 11β-HSD1 is enzymatically coupled to 17β-HSD3, utilizing NADPH and NADP in intermeshed regeneration systems.     

Characterization of the InsP6-dependent interaction between CK2 and Nopp140

Nopp140, a highly phosphorylated nucleolar protein, negatively regulates CK2, a kinase essential for cell proliferation. We quantitatively analyzed the interaction between two subunits of CK2 and Nopp140 and characterized the mechanism by which InsP₆ inhibits the interaction. Nopp140 specifically binds to the catalytic subunit of CK2 (CK2α) with a dissociation constant of (K_d) of 4 nM, which interferes with the catalytic activity of CK2. The C-terminal region of Nopp140 is determined as CK2α-binding region by a yeast two-hybrid method as well as a direct measurement of the interaction between CK2α and deletion mutants of Nopp140. InsP₆ specifically binds to CK2α and disrupts the interaction between CK2α and Nopp140 with an IC₅₀ value of 25 μM, thereby attenuating the Nopp140-mediated repression of CK2 activity.     

Pharmacological evidence that Ca²+ channels and, to a lesser extent, K+ channels mediate the relaxation of testosterone in the canine basilar artery

Testosterone induces vasorelaxation through non-genomic mechanisms in several isolated blood vessels, but no study has reported its effects on the canine basilar artery, an important artery implicated in cerebral vasospasm. Hence, this study has investigated the mechanisms involved in testosterone-induced relaxation of the canine basilar artery. For this purpose, the vasorelaxant effects of testosterone were evaluated in KCl- and/or PGF_2α-precontracted arterial rings in vitro in the absence or presence of several antagonists/inhibitors/blockers; the effect of testosterone on the contractile responses to CaCl₂ was also determined. Testosterone (10-180 μM) produced concentration-dependent relaxations of KCl- or PGF_2α-precontracted arterial rings which were: (i) unaffected by flutamide (10 μM), dl-aminoglutethimide (10 μM), actinomycin D (10 μM), cycloheximide (10 μM), SQ 22,536 (100 μM) or ODQ (30 μM); and (ii) significantly attenuated by the blockers 4-aminopyridine (K_V; 1 mM), BaCl₂ (K_IR; 30 μM), iberiotoxin (BKCa2+; 20 nM), but not by glybenclamide (K_ATP; 10 μM). In addition, testosterone (31, 56 and 180 μM) and nifedipine (0.01-1 μM) produced a concentration-dependent blockade of the contraction to CaCl₂ (10 μM to 10 mM) in arterial rings depolarized by 60 mM KCl. These results, taken together, show that testosterone relaxes the canine basilar artery mainly by blockade of voltage-dependent Ca²⁺ channels and, to a lesser extent, by activation of K⁺ channels (K_IR, K_V and BKCa2+). This effect does not involve genomic mechanisms, production of cAMP/cGMP or the conversion of testosterone to 17β-estradiol.     

Pseudohypericin is necessary for the light-activated inhibition of prostaglandin E2 pathways by a 4 component system mimicking an Hypericum perforatum fraction

Hypericum perforatum (Hp) has been used medicinally to treat a variety of conditions including mild-to-moderate depression. Recently, several anti-inflammatory activities of Hp have been reported. An ethanol extract of Hp was fractionated with the guidance of an anti-inflammatory bioassay (lipopolysaccharide (LPS)-induced prostaglandin E₂ production (PGE₂)), and four constituents were identified. When combined together at concentrations detected in the Hp fraction to make a 4 component system, these constituents (0.1 μM chlorogenic acid (compound 1), 0.08 μM amentoflavone (compound 2), 0.07 μM quercetin (compound 3), and 0.03 μM pseudohypericin (compound 4)) explained the majority of the activity of the fraction when activated by light, but only partially explained the activity of this Hp fraction in dark conditions. One of the constituents, light-activated pseudohypericin, was necessary, but not sufficient to explain the reduction in LPS-induced PGE₂ of the 4 component system. The Hp fraction and the 4 component system inhibited lipoxygenase and cytosolic phospholipase A₂, two enzymes in the PGE₂-mediated inflammatory response. The 4 component system inhibited the production of the pro-inflammatory cytokine tumor necrosis factor-α (TNF-α), and the Hp fraction inhibited the anti-inflammatory cytokine interleukin-10 (IL-10). Thus, the Hp fraction and selected constituents from this fraction showed evidence of blocking pro-inflammatory mediators but not enhancing inflammation-suppressing mediators.     

The effects of all-trans retinoic acid on estrogen receptor signaling in the estrogen-sensitive MCF/BUS subline

Estrogen receptor alpha (ERα) and retinoic acid receptors (RARs) play important and opposite roles in breast cancer growth. While exposure to ERα agonists such as 17β-estradiol (E₂) is related to proliferation, RAR agonists such as all-trans retinoic acid (AtRA) induce anti-proliferative effects. Although crosstalk between these pathways has been proposed, the molecular mechanisms underlying this interplay are still not completely unraveled. The aim of this study was to evaluate the effects of AtRA on ERα-mediated signaling in the ERα positive cell lines MCF7/BUS and U2OS-ERα-Luc to investigate some of the possible underlying modes of action. To do so, this study assessed the effects of AtRA on different ERα-related events such as ERα-mediated cell proliferation and gene expression, ERα-coregulator binding and ERα subcellular localization. AtRA-mediated antagonism of E₂-induced signaling was observed in the proliferation and gene expression studies. However, AtRA showed no remarkable effects on the E₂-driven coregulator binding and subcellular distribution of ERα. Interestingly, in the absence of E₂, ERα-mediated gene expression, ERα-coregulator binding and ERα subcellular mobilization were increased upon exposure to micromolar concentrations of AtRA found to inhibit cell proliferation after long-term exposure. Nevertheless, experiments using purified ERα showed that direct binding of AtRA to ERα does not occur. Altogether, our results using MCF7/BUS and U2OS-ERα-Luc cells suggest that AtRA, without being a direct ligand of ERα, can indirectly interfere on basal ERα-coregulator binding and basal ERα subcellular localization in addition to the previously described crosstalk mechanisms such as competition of ERs and RARs for DNA binding sites.     

Involvement of diacylglycerol produced by phospholipase D activation in Aβ-induced reduction of sAPPα secretion in SH-SY5Y neuroblastoma cells

We previously reported that the thiol proteinase inhibitor, E-64-d, ameliorated amyloid β (Aβ)-induced reduction of soluble amyloid precursor protein α (sAPPα) secretion by reversing ceramide-induced protein kinase C down-regulation in SH-SY5Y neuroblastoma cells. In the present study, we showed that Aβ (1–42) peptide enhanced diacylglycerol (DAG) production by phospholipase D (PLD) activation in these cells. We subsequently examined whether PLD was involved in Aβ-induced reduction of sAPPα secretion and showed that 2 μM CAY10593, which selectively inhibits PLD2, ameliorated reduction of sAPPα secretion, whereas 50 nM CAY10593, which selectively inhibits PLD1, did not. Moreover, 50 µM propranolol, a phosphatidic acid phosphohydrolase inhibitor, also ameliorated Aβ-induced reduction of sAPPα secretion, suggesting that DAG may be responsible for Aβ-induced reduction of sAPPα. We subsequently examined whether DAG affects sAPPα secretion and showed that a DAG analog reduced sAPPα secretion in SH-SY5Y cells. In addition, DAG enhanced ceramide production by stimulating neutral sphingomyelinase (N-SMase) activity. We previously demonstrated that Aβ stimulates N-SMase activity in SH-SY5Y cells. Here, we showed that inhibition of PLD2 by 2 μM CAY10593 suppressed Aβ-induced N-SMase activation. Taken together, the results suggest that DAG produced through the PLD pathway is involved in Aβ-induced reduction of sAPPα secretion in SH-SY5Y cells.