Inhibitory Effect Of Estradiol Research Articles

Hormonal Underpinnings of the Variation in Sexual Desire, Arousal and Activity Throughout the Menstrual Cycle – A Multifaceted Approach

ABSTRACT Although prior evidence supports women’s mating behaviors and preferences being related to ovarian hormonal levels, there is conflicting evidence about exactly which hormones predict sexual function best, which specific psychosexual facets are affected and how between-individual and within-individual differences relate to this question. In this study levels of estradiol and progesterone were measured (once daily for 15 days for each participant) for 97 women, who attended two testing sessions, in times of the cycle varying in conception probability (based on the luteinizing hormone (LH) test result). Women completed surveys on their sexual desire, arousal, sexual activity frequency and initiation. There was a significant difference between peri-ovulatory and luteal values for all sexual function variables. Between-subject progesterone negatively predicted sexual activity frequency only. Within-subject estradiol positively and progesterone negatively predicted sexual desire. The findings provide support for hormonal underpinnings of sexual desire and sexual activity frequency fluctuations during the menstrual cycle. The findings did not yield support for hormonal influences on sexual arousal and initiation of sexual encounters. The main findings are consistent with the excitatory and inhibitory effects of estradiol and progesterone, respectively, on measures of women’s sexual motivation.

Estradiol inhibits HIV-1BaL infection and induces CFL1 expression in peripheral blood mononuclear cells and endocervical mucosa

An inhibitory effect of estradiol (E2) on HIV-1 infection was suggested by several reports. We previously identified increased gene expression of actin-binding protein cofilin 1 (CFL1) in endocervix in the E2-dominated proliferative phase of the menstrual cycle. Actin cytoskeleton has an integral role in establishing and spreading HIV-1 infection. Herein, we studied in vitro effects of E2 on HIV-1 infection and on CFL1 expression to gain insight into the mechanism of HIV-1 inhibition by E2. E2 dose-dependently inhibited HIV-1BaL infection in peripheral blood mononuclear cells (PBMCs) and endocervix. In PBMCs and endocervix, E2 increased protein expression of total CFL1 and phosphorylated CFL1 (pCFL1) and pCFL1/CFL1 ratios. LIMKi3, a LIM kinase 1 and 2 inhibitor, abrogated the phenotype and restored infection in both PBMCs and endocervix; inhibited E2-induced expression of total CFL1, pCFL1; and decreased pCFL1/CFL1 ratios. Knockdown of CFL1 in PBMCs also abrogated the phenotype and partially restored infection. Additional analysis of soluble mediators revealed decreased concentrations of pro-inflammatory chemokines CXCL10 and CCL5 in infected tissues incubated with E2. Our results suggest a link between E2-mediated anti-HIV-1 activity and expression of CFL1 in PBMCs and endocervical mucosa. The data support exploration of cytoskeletal signaling pathway targets for the development of prevention strategies against HIV-1.

Bidirectional effects of estradiol on the control of water intake in female rats

The inhibitory effect of estradiol (E2) on water intake has been recognized for 50 years. Despite a rich literature describing this phenomenon, we report here a previously unidentified dipsogenic effect of E2 during states of low fluid intake. Our initial goal was to test the hypothesis that the anti-dipsogenic effect of E2 on unstimulated water intake is independent of its anorexigenic effect in female rats. In support of this hypothesis, water intake was reduced during estrus, compared to diestrus, when food was present or absent. Water intake was reduced by E2 in ovariectomized rats when food was available, demonstrating a causative role of E2. Surprisingly, however, when food was removed, resulting in a significant reduction in baseline water intake, E2 enhanced drinking. Accordingly, we next tested the effect of E2 on water intake after an acute suppression of intake induced by exendin-4. The initial rebound drinking was greater in E2-treated, compared to Oil-treated, rats. Finally, to reconcile conflicting reports regarding the effect of ovariectomy on water intake, we measured daily water and food intake, and body weight in ovariectomized and sham-operated rats. Predictably, ovariectomy significantly increased food intake and body weight, but only transiently increased water intake. Together these results provide further support for independent effects of E2 on the controls of water and food intake. More importantly, this report of bidirectional effects of E2 on water intake may lead to a paradigm shift, as it challenges the prevailing view that E2 effects on fluid intake are exclusively inhibitory.

Estradiol via estrogen receptor beta inhibits chondrogenesis of mouse vertebral growth plate in vitro.

Abnormal growth of vertebral growth plate (VGP) was considered as one of the etiologic factors in adolescent idiopathic scoliosis (AIS). Previous studies described that estrogen played an important role in the pathogenesis of AIS. The present study was aimed to investigate the effect of estrogen/estrogen receptor axis on mouse VGP chondrocytes in vitro. Chondrocytes were isolated from mouse VGP and treated with or without 17β-estradiol (E2). Cell proliferation was measured by the cell growth rate assay. Gene expression of collagen type II and aggrecan were evaluated by real-time PCR. Expression of the proliferating cell nuclear antigen (PCNA), Sox9, and Smad4 were detected by Western blotting. Estradiol inhibited the proliferation of VGP chondrocytes and the gene expression of collagen type II and aggrecan and downregulated the protein expression of PCNA, Sox9, and Smad4. In addition, the inhibitory effect of estradiol was reversed by ERβ small interfering RNA (siRNA) or PHTPP, an ERβ antagonist. Estradiol via estrogen/estrogen receptor β axis inhibits the proliferation and differentiation of VGP chondrocytes, which might give some new insight into the regulatory mechanism of bone development.

Estradiol receptors mediate estradiol-induced inhibition of mitochondrialCa2+ efflux in rat caudate nucleus and brain stem

Our earlier studies found that in vitro estradiol modulates mitochondrial Ca2+ transport in discrete brain regions. The present study examined the role of estradiol receptors (ERs) in estradiol-induced inhibition of Ca^{2+} efflux from synaptosomal mitochondria isolated from rat caudate nuclei and brain stems. Radioactively labeled CaCl_2 (0.6?0.75 µCi ^45CaCl_{2}) was used for Ca^{2+} transport monitoring. The results revealed that in the presence of ER antagonist 7\alpha,17AŸ-[9[(4,4,5,5,5-pentafluoropentyl)sulfinyl]nonyl]estra-1,3,5(10)-triene-3,17-diol (ICI 182,780) (1 µmol/L), the inhibitory effect of estradiol on mitochondrial Ca^{2+} efflux was more than 60% decreased, suggesting the involvement of ER in this mode of estradiol neuromodulatory action. When particular contributions of ER\alpha and ERAŸ were tested, it was found that ERAŸ agonist 2,3-bis(4-hydroxy phenyl)-propionitrile (10 nmol/L) inhibited Ca^{2+} efflux more than 20%, while the inhibition with ER\alpha agonist 4,4?,4??-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (10 nmol/L) was about 10%, both compared to the control. Both agonists demonstrated attenuation of Ca^{2+} efflux decrease in the presence of mitochondrial Na^{+}/Ca^{2+} exchanger antagonist 7-chloro-5-(2-chlorophenyl)-1,5-dihyhdro-4,1-benzothiazepin-2(3H)-one (10 µmol/L), showing interference with the inhibitory action of that agent. Our results strongly indicate ERs as the mediators of estradiol-induced mitochondrial Ca^{2+} efflux inhibition in rat caudate nucleus and brain stem synaptosomes.

Cyclic estradiol treatment modulates the orexigenic effects of ghrelin in ovariectomized rats

Data from a wide variety of mammalian species indicate that feeding behavior can be influenced by changes in endogenous estrogens and exogenous estrogenic treatments. Ghrelin is an important physiological signal for the regulation of energy balance, and ghrelin treatment increases eating and body weight in male rodents. The following studies evaluated the hypothesis that the inhibitory effects of estradiol on feeding involve interactions with orexigenic peptides by examining the ability of estradiol to modulate the behavioral effects of ghrelin in female rats. In these experiments, adult rats were ovariectomized and assigned to an estradiol benzoate (EB) or an oil (control) group. Three weeks after ovariectomy, animals received two daily subcutaneous injections of EB or the oil vehicle. Animals then received intraperitoneal (ip) injections of ghrelin (6.0 or 12.0nmol) or saline during the nocturnal and diurnal periods three days after the first injection of estradiol or oil. Food intake, meal size, and meal number were determined during the 2-hour period following ghrelin or saline treatments. Ghrelin significantly increased food intake during nocturnal tests in oil-treated but not estradiol-treated rats. The hyperphagic effects of ghrelin on nocturnal food intake were also accompanied by an increase in meal size, and this effect of ghrelin on meal size was attenuated in estradiol-treated females. These findings support the hypothesis that the effects of estradiol on feeding behavior involve an attenuation of orexigenic signals, possibly by modulating the effects of the peripheral ghrelin signal on hypothalamic neuropeptides involved in the control of food intake.

Estradiol Regulation of Constitutive and Keratinocyte Growth Factor-Induced CCL20 and CXCL1 Secretion by Mouse Uterine Epithelial Cells

Estradiol can directly affect epithelial cells or indirectly affect epithelial cells via stromal fibroblast secretion of growth factors, such as keratinocyte growth factor (KGF). The purpose of the present study was to determine whether estradiol regulates constitutive as well as KGF-induced uterine epithelial cell secretion of CCL20 and CXCL1. Freshly isolated and polarized uterine epithelial cells from Balb/c mice were cultured with estradiol in the presence or absence of KGF. CCL20 and CXCL1 were measured by ELISA. Estradiol inhibited CCL20 secretion by freshly isolated and polarized uterine epithelial cells in the presence or absence of KGF. Unexpectedly, it enhanced KGF-induced CXCL1 secretion beyond that seen with KGF alone. Estradiol increased CXCL1 secretion at 24 hr and inhibited CCL20 at 48 hr. The effects of estradiol are specific in that progesterone, cortisol, dihydrotestosterone, and aldosterone had no effect on either CCL20 or CXCL1 secretion. The inhibitory effect of estradiol on CCL20 secretion was reversed with ICI 182,780, an estrogen receptor antagonist, indicating that this effect is estrogen receptor mediated. Our data indicate that estradiol is important in regulating the effects of KGF on mouse uterine epithelial cell secretion of CCL20 and CXCL1.

TRIENNIAL REPRODUCTION SYMPOSIUM: The ovarian follicular reserve in cattle: What regulates its formation and size?1,2

The ovarian follicular reserve has been linked to fertility in cattle. Young adult cattle with low vs. high numbers of antral follicles ≥ 3 mm in diameter in follicular waves also have fewer preantral follicles and decreased fertility. This underscores the importance of understanding the factors that regulate early follicular development and establish the ovarian follicular reserve, but little is known about how the follicular reserve is first established. In ruminants and humans, follicles form during fetal life, but there is a gap (about 50 d in cattle) between the appearance of the first primordial follicles and the first growing, primary follicles. In this review we present evidence that in cattle, fetal ovarian steroids (i.e., estradiol and progesterone) are negative regulators of both follicle formation and of the acquisition by newly formed follicles of the capacity to activate (i.e., initiate growth). The results indicate that capacity to activate is linked to the completion of meiotic prophase I by the oocyte. The inhibitory effects of estradiol on follicle activation were found to be reversible and correlated with inhibition of the progression of meiotic prophase I. Fetal bovine ovaries produce steroid hormones and production varies considerably during gestation and in a pattern consistent with the hypothesis that they inhibit follicle formation and capacity of newly formed follicles to activate in vivo. However, little was known about how steroid production is regulated. In our studies, both LH and FSH stimulated progesterone and estradiol production by ovarian pieces in vitro. The addition of testosterone to the culture medium enhanced estradiol production, especially when FSH was also present, but inhibited progesterone production, even in the presence of gonadotropins. Evidence is also presented for effects of maternal nutrition and health and for potential effects of estrogenic endocrine-disrupting chemicals on the size of the ovarian follicular reserve established during fetal life. In summary, fetal ovarian steroids may be important regulators of the early stages of follicular development in cattle. Therefore, external factors that alter steroid production or action may affect the size of the ovarian follicular reserve.

Protective Role of Estrogen-induced miRNA-29 Expression in Carbon Tetrachloride-induced Mouse Liver Injury

Previous studies have indicated that female animals are more resistant to carbon tetrachloride (CCl(4))-induced liver fibrosis than male animals, and that estradiol (E(2)) treatment can inhibit CCl(4)-induced animal hepatic fibrosis. The underlying mechanism governing these phenomena, however, has not been fully elucidated. Here we reported the role of estrogen-induced miRNA-29 (miR-29) expression in CCl(4)-induced mouse liver injury. Hepatic miR-29 levels were differentially regulated in female and male mice during CCl(4) treatment. Specifically, the levels of miR-29a and miR-29b expression were significantly decreased in the livers of male, but not female, mice following 4 weeks of CCl(4) treatment. The down-regulation of miR-29a and miR-29b in male mouse livers correlated with the early development of liver fibrosis, as indicated by increased expressions of fibrotic markers in male mice relative to female mice. In addition, E(2) was maintained at a higher level in female mice than in male mice. In contrast to TGF-β1 that decreased miR-29a/b expression in murine hepatoma IAR20 cells and normal hepatocytes, E(2) enhanced the expression of miR-29a/b through suppression of the nuclear factor-κB (NF-κB) signal pathway, which negatively regulates miR-29 expression. Furthermore, both E(2) treatment and intravenous injection of the recombinant adenovirus expressing miR-29a/b markedly increased the miR-29a/b level and attenuated the expression of fibrotic markers in mouse livers during CCl(4) treatment, supporting the protective role of E(2)-induced miR-29 in CCl(4)-induced hepatic injury. In conclusion, our results collectively demonstrate that estrogen can inhibit CCl(4)-induced hepatic injury through the induction of hepatic miR-29.

Effect of oestrogen on T cell apoptosis in patients with systemic lupus erythematosus

Defective control of T cell apoptosis is considered to be one of the pathogenetic mechanisms in systemic lupus erythematosus (SLE). Oestrogen has been known to predispose women to SLE and also to exacerbate activity of SLE; however, the role of oestrogen in the apoptosis of SLE T cells has not yet been documented. In this study, we investigated the direct effect of oestrogen on the activation-induced cell death of T cells in SLE patients. The results demonstrated that oestradiol decreased the apoptosis of SLE T cells stimulated with phorbol 12-myristate 13-acetate (PMA) plus ionomycin in a dose-dependent manner. In addition, oestradiol down-regulated the expression of Fas ligand (FasL) in activated SLE T cells at the both protein and mRNA levels. In contrast, testosterone increased FasL expression dose-dependently in SLE T cells stimulated with PMA plus ionomycin. The inhibitory effect of oestradiol on FasL expression was mediated through binding to its receptor, as co-treatment of tamoxifen, an oestrogen receptor inhibitor, completely nullified the oestradiol-induced decrease in FasL mRNA expression. Moreover, pre-treatment of FasL-transfected L5178Y cells with either oestradiol or anti-FasL antibody inhibited significantly the apoptosis of Fas-sensitive Hela cells when two types of cells were co-cultured. These data suggest that oestrogen inhibits activation-induced apoptosis of SLE T cells by down-regulating the expression of FasL. Oestrogen inhibition of T cell apoptosis may allow for the persistence of autoreactive T cells, thereby exhibiting the detrimental action of oestrogen on SLE activity.

Effects of estradiol on food intake and meal patterns for diets that differ in flavor and fat content

Apart from the well known inhibitory effects of estradiol on food intake, meal size, and body weight in female rats that have been documented over the past thirty years, a more recent report presents the opposite finding; that a large dose of estradiol can increase food intake and weight gain in gonadally intact female rats presented with a palatable diet. The purpose of the present experiment was to further examine this hypothesis by evaluating the ability of estradiol to influence feeding behavior in ovariectomized rats presented with diets that differ in flavor and fat content. Female rats were given a cyclic regimen of estradiol benzoate treatment (5.0 or 20.0 µg) or the oil vehicle and were presented with the standard chow diet or a diet with a higher fat content and chocolate flavor. Food intake, meal size, and meal number were monitored three days after the first injection of estradiol or oil. Compared to the chow diet, food intake increased when animals had access to the chocolate/fat diet during the vehicle treatment condition. Both doses of estradiol significantly decreased food intake, meal size, and body weight gain when animals were presented with either the standard chow diet or the chocolate/fat diet. These findings indicate that estradiol does not stimulate the intake of a palatable diet in ovariectomized rats, and suggest that previous results showing that estradiol enhanced eating and weight gain stemmed from a disruption of the hypothalamic–pituitary–gonadal axis when intact females received a large dose of exogenous estradiol.

Inhibition of Activation of Newly Formed Bovine Primordial Follicles by Estradiol Is Reversible and Associated with Inhibition of Progression of Meiotic Prophase I.

In some mammals, like cattle, sheep, and humans, the pool of resting, primordial follicles is formed during fetal life and little is known about the regulation of the early stages of ovarian follicular development. Bovine primordial and primary follicles first appear around day 90 and 140 of gestation, respectively (gestation length = 280 days). Estradiol inhibits activation (i.e. initiation of growth) of newly formed bovine primordial follicles in vitro and secretion of estradiol by bovine fetal ovaries from around 83 to 140-day-old fetuses declines precipitously with age, suggesting an inhibitory role for estradiol in follicle activation in vivo. If inhibition of activation of newly formed bovine primordial follicles by estradiol in vitro mimics a normal developmental mechanism, we would expect the inhibition to be reversible. Therefore, the first objective of the present study was to test the hypothesis that the inhibitory effect of estradiol on follicle activation is reversible. Pieces of fetal ovarian cortex (4 pieces/treatment/fetus; n = 2 fetuses; day 100 and 120 of gestation) were cultured in serum-free medium supplemented with ITS+ (control medium) for 10 or 14 days (controls), with estradiol (10-6 M) for 10 days, or with estradiol for 10 days and then in control medium for 4 more days. On day 0 (time 0 controls) and after culture, cortical pieces were fixed and embedded in paraffin for serial sectioning and sections were stained with H&E for morphmetric analysis. On day 0, cortical pieces contained mostly primordial follicles (99.95%), whereas after culture the percentages of primary follicles in all cultures, except cultures treated with estradiol, were significantly higher than in day 0 controls (P < 0.05), suggesting that follicle activation occurred in vitro. There was a higher percentage of primary follicles in day 14 controls than in day 10 controls (75.4 ± 4% vs. 41.7 ± 8.3%; P < 0.05), suggesting that the number of follicles capable of activating increased with time in culture. The percentage of primary follicles in cortical pieces cultured with estradiol was lower compared with day 10 or 14 controls, (12 ± 4.6 vs. 41.7 ± 8.3 and 75.4 ± 4%, respectively; P < 0.05), consistent with our previous results showing that estradiol inhibits follicle activation. The inhibitory effects of estradiol on activation were reversed by culturing cortical pieces in control medium from day 10-14 (12 ± 4.6 vs. 66.9 ± 13.8% primary follicles; P < 0.05). Since we showed previously that the capacity of bovine primordial follicles to activate is associated with the achievement of meiotic arrest in their oocytes at the diplotene stage of prophase I, we next examined whether the inhibition of follicle activation by estradiol is associated with inhibition of the progression of meiotic prophase I. The results showed a higher percentage of primordial follicles with oocytes in pre-meitotic arrest (pre-diplotene) stages in cultures treated with estradiol than in day 10 or day 14 controls (92.5 ± 7 vs. 43.2 ± 5 or 13.5 ± 13, respectively; P < 0.05). In summary, the capacity of newly formed bovine primordial follicles to activate increases with time in culture. Inhibition of activation of newly formed bovine follicles by estradiol is reversible and is associated with inhibition of the progression of meiotic prophase I.

Identification of Non-Genomic Mechanisms for Estrogen Effects on Monocyte Responses to Inflammatory and Osteoclastogenic Stimuli.

In previous studies we found that estradiol can act directly on primary human monocytes to inhibit RANKL stimulated osteoclastic differentiation, a potential mechanism for anti-osteoporotic effects of estrogen. Estrogen effects were rapid, suggesting involvement of non-genomic mechanisms in the inhibition of osteoclastogenic signals. Direct effects of estradiol on monocytic cells have also been implicated in anti-inflammatory effects of estrogen. It was recently shown that in murine monocytic cells, estrogen could acutely inhibit signaling events downstream from the lipopolysaccharide receptor, Toll-like receptor (TLR) 4 (Ghisletti et al. Mol Cell Biol 25 (2005) 2957). A critical component of TLR effects is the activation of the transcription factor NF-kB, resulting in increased expression of cytokines and chemokines that initiate the inflammatory response to LPS and other stimuli. NF-kB is also a critical mediator of the effects of TNF family members including RANKL. RANKL differs from most TNF family members in relying primarily on a signaling pathway through Traf6 for activation of NF-kB. Traf6 is also a key component of the pathway from TLR to NF-kB, and signaling events downstream from Traf6 appear to be common to RANKL and TLR4 signaling. We used CD14-selected primary human monocytes to examine the role of estrogen effects on NF-kB in its inhibition of monocyte response to RANKL and to determine whether common molecular mechanisms underlie estrogen effects on inflammation and osteoclastogenesis. Estradiol was added to CD14 cell cultures that, after 5 minutes, were treated with RANKL or lipopolysaccharide. After 30 minutes, cells were fixed and NF-kB localization determined by immunofluorescence microscopy using antibodies to p65. The nuclear translocation of NF-kB, evident in parallel cultures treated with RANKL or LPS but not estrogen, was markedly inhibited when cells were cotreated with estradiol. (Estrogen alone showed no effect on NF-kB localization.) Alternatively, after treatment with or without estrogen, LPS or RANKL, nuclear and cytosolic extracts were prepared and evaluated by SDS-PAGE and DNA binding, to confirm estrogen inhibition of the nuclear translocation of NF-kB. Nuclear localization of NF-kB follows the phosphorylation and degradation of IkB. Cells stimulated with RANKL or LPS for 5 or 20 minutes showed inhibition of IkB degradation if cells were cotreated with estradiol. This suggested involvement of the kinase cascade initiated by a signaling protein complex including Traf6, which is the most proximal signaling protein common to the TLR and RANKL pathways. We investigated the possibility that estrogen inhibited TLR and RANKL signal transduction through effects on the Traf6 protein complex. After 5 minutes stimulation with ligands, coimmunoprecipitation studies revealed the association of Traf6 with estrogen receptor alpha (ER) when cells were cotreated with estradiol. Coimmunoprecipitation further revealed the association of the scaffolding protein BCAR1 (p130 Cas), a protein previously implicated in non-genomic estrogen effects in breast cancer cells. We evaluated the role of BCAR1 using siRNA to inhibit its expression. In cells lacking BCAR1, the inhibitory effect of estradiol on NF-kB nuclear translocation was abrogated. The results overall suggest that estradiol may inhibit both TLR and RANKL signaling through ER protein interactions, potentially mediated by BCAR1, that disrupt the Traf6 signaling complex critical for activation of NF-kB.

Oestrogen formation and connective tissue growth factor expression in rat granulosa cells

Ovarian follicular development involves continual remodelling of the extracellular matrix (ECM) forming the basement membrane and intercellular framework that support granulosa cell (GC) growth and differentiation. Insight into the molecular regulation of ovarian ECM remodelling is potentially translatable to tissue remodelling elsewhere in the body. We therefore studied the link between a gene marker of ECM remodelling (connective tissue growth factor (CTGF)) and oestrogen biosynthesis (cytochrome P450(aromatase) (P450(arom))) in rat granulosa cells. To determine if a cause-effect interaction exists, we used semi-quantitative in situ hybridisation to analyse patterns of CTGF and P450(arom) mRNA expression and immunohistochemistry to detect CTGF protein localisation throughout follicular development, and tested the actions of CTGF on oestrogen biosynthesis and oestradiol on CTGF mRNA expression in isolated GC in vitro. CTGF mRNA levels in GC rose gradually through small preantral (SP) and small antral (SA) stages of development to a maximum (fivefold higher) in large antral (LA) follicles. In preovulatory (PO) follicles, the CTGF mRNA level fell to 30% of that in SP follicles. P450(arom) mRNA also increased (threefold in LA relative to SP) throughout antral development follicles, but in contrast to CTGF continued to increase (12-fold) in PO follicles. In the cumulus oophorus of PO follicles, the residual GC CTGF mRNA expression increased with proximity to the oocyte, being inversely related to P450(arom). Elsewhere in the follicle wall, there was a mural-to-antral gradient of CTGF mRNA expression, again inversely related to P450(arom). Immunohistochemistry showed CTGF protein localisation broadly followed mRNA expression during follicular development, although the protein was also present in the theca interna and ovarian surface epithelium. Gradients in CTGF expression across the cumulus oophorus and follicle wall were similar to those observed for mRNA with CTGF protein expression being greatest in proximity to the oocyte. Treatment of isolated GC from preantral and SA follicles with recombinant human CTGF (1-100 ng/ml) did not affect basal or FSH-stimulated GC aromatase activity. However, in the absence of FSH, oestradiol (10(-7)-10(-5) M) stimulated CTGF mRNA expression up to twofold. Conversely, FSH (10 ng/ml) alone reduced CTGF mRNA expression by 40% and combined treatment with FSH and oestradiol further suppressed CTGF to 10% of the control value. The oestrogen receptor (ER) antagonist, ICI 182 780 blocked the stimulatory and inhibitory effects of oestradiol, suggesting a specific ER-mediated mode of action on CTGF. Therefore, CTGF gene expression in GC is under local control by oestrogen whose effect (positive or negative) is modulated by FSH. This helps explain why gene expression of CTGF and P450(arom) diverge in FSH-induced PO follicles and has implications for oestrogenic regulation of CTGF formation elsewhere in the body.

IL-1β-Mediated Proinflammatory Responses Are Inhibited by Estradiol via Down-Regulation of IL-1 Receptor Type I in Uterine Epithelial Cells

The objective of this study was to examine the effects of sex hormones on IL-1beta-mediated responses by uterine epithelial cells. The mRNA expression and secretion of human beta-defensin-2 and CXCL8 by uterine epithelial cells was examined following stimulation with IL-1beta in the presence of estradiol or progesterone. Estradiol inhibited the IL-1beta-mediated mRNA expression and secretion of human beta-defensin-2 and CXCL8 by uterine epithelial cells while progesterone had no effect. Inhibition of the IL-1beta-mediated response by estradiol was dose dependent, with maximal inhibition observed using 10(-7) to 10(-10) M, and was shown to be mediated through the estrogen receptor because addition of a pure estrogen receptor antagonist abrogated this effect. The mechanism by which estradiol inhibits IL-1beta-mediated responses by uterine epithelial cells appears to be the down-modulation of the IL-1R type I, thereby reducing the uterine epithelial cell's ability to respond to IL-1beta. These results suggest that the inhibitory effect of estradiol on IL-1beta-mediated inflammatory responses by uterine epithelial cells indicates a link between the endocrine and immune systems and may be crucial for dampening proinflammatory responses during the time of ovulation or pregnancy.

Membranal effects of phytoestrogens and carboxy derivatives of phytoestrogens on human vascular and bone cells: New insights based on studies with carboxy-biochanin A

Estradiol-17β (E 2) and some phytoestrogens induce a biphasic effect on DNA synthesis in cultured human vascular smooth muscle cells (VSMC), i.e., stimulation at low concentrations and inhibition at high concentrations. These compounds also increase the specific activity of creatine kinase (CK) as well as intracellular Ca 2+ concentration in both VSMC and human female-derived cultured bone cells (OBs), and stimulate ERK1/2 phosphorylation in VSMC. At least some of these effects are exerted via membranal binding sites (mER), as would appear from observations that protein-bound, membrane impermeant estrogenic complexes can mimic the effect of E 2 on DNA synthesis, intracellular Ca 2+ concentration and MAPK, but not on CK activity. We now extend these studies by examining the effects of a novel carboxy-derivative of biochanin A, 6-carboxy-biochanin A (cBA) in VSMC and human osteoblasts in culture. cBA increased DNA synthesis in VSMC in a dose-dependent manner and was able to maintain this effect when linked to a cell membrane impermeable protein. In VSMC both cBA and estradiol, in their free or protein-bound forms induced a steep and immediate rise in intracellular calcium. Both the free and protein-bound conjugates of cBA and estradiol increased net MAPK-kinase activity. Neither the stimulatory effect of cBA nor the inhibitory effect of estradiol on DNA synthesis in VSMC could be shown in the presence of the MAPK-kinase inhibitor UO126. The presence of membrane binding sites for both estradiol and cBA was supported by direct visualization, using fluorescence labeling of their respective protein conjugates, E 2-BSA and cBA-ovalbumin. Furthermore, these presumed membrane ER for estradiol and cBA were co-localized. In cultured human osteoblasts, cBA stimulated CK activity in a dose related fashion, which paralleled the increase in CK induced by estradiol per se, confirming the estrogenic properties of cBA in human bone cells. Both the free and protein-bound forms of cBA elicited immediate and substantial increments in intracellular Ca 2+, similar to, but usually larger than the responses elicited by estradiol per se. cBA also increased ERα and suppressed ERβ mRNA expression in human osteoblasts. Cultured human osteoblasts also harbor membrane binding sites for protein-bound form of cG, which are co-localized with the binding sites for protein-bound estradiol. The extent to which these properties of the novel synthetic phytoestrogen derivatives may be utilized to avert human vascular and/or bone disease requires further study.

CYP450- and COMT-derived estradiol metabolites inhibit activity of human coronary artery SMCs.

The purpose of this study is to test the hypothesis that the inhibitory effects of estradiol in human coronary vascular smooth muscle cells are mediated via local conversion to methoxyestradiols via specific cytochrome P450s (CYP450s) and catechol-O-methyltransferase (COMT). The inhibitory effects of estradiol on serum-induced cell activity (DNA synthesis, cell number, collagen synthesis, and cell migration) were enhanced by 3-methylcholantherene, phenobarbital (broad-spectrum CYP450 inducers), and beta-naphthoflavone (CYP1A1/1A2 inducer) and were blocked by 1-aminobenzotriazole (broad-spectrum CYP450 inhibitor). Ellipticine, alpha-naphthoflavone (selective CYP1A1 inhibitors), and pyrene (selective CYP1B1 inhibitor), but not ketoconazole (selective CYP3A4 inhibitor) or furafylline (selective CYP1A2 inhibitor), abrogated the inhibitor effects of estradiol on cell activity, a profile consistent with a CYP1A1/CYP1B1-mediated mechanism. The inhibitory effects of estradiol were blocked by the COMT inhibitors OR486 and quercetin. The estrogen receptor antagonist ICI 182,780 blocked the inhibitory effects of estradiol, but only at concentrations that also blocked the metabolism of estradiol to hydroxyestradiols (precursors of methoxyestradiols). Western blot analysis revealed that coronary smooth muscle cells expressed CYP1A1 and CYP1B1. Moreover, these cells metabolized estradiol to hydroxyestradiols and methoxyestradiols, and the conversion of 2-hydroxyestradiol to 2-methoxyestradiol was blocked by OR486 and quercetin. These findings provide evidence that the inhibitory effects of estradiol on coronary smooth muscle cells are largely mediated via CYP1A1- and CYP1B1-derived hydroxyestradiols that are converted to methoxyestradiols by COMT.

Estrogen receptor beta is involved in the anorectic action of estrogen.

Estrogen has been implicated in feeding behavior and adiposity. This study was undertaken to elucidate the mechanism underlying the anti-obesity and anorectic action of estrogen and the role of estrogen receptor (ER) in the central nervous system. Ovariectomy in 8-week-old female Wistar rats induced hyperphagia along with an increase in body weight and abdominal fat accumulation compared to control sham-operated rats. These changes were fully reversed by subcutaneous replacement of estradiol and were abrogated by pair-feeding. Then, the effects of intracerebroventricular infusion of estradiol, alone or in combination with antisense oligodeoxynucleotides (ODN), for ER in ovariectomized rats were examined. The estradiol group showed 10-20% lower daily food intake, and after the 2-week infusion period a 14% reduction in body weight with a similar reduction in abdominal fat compared to the vehicle group. The inhibitory effect of estradiol on food intake and body weight was blocked by co-administration of ER-beta antisense ODN, whereas ER-alpha antisense ODN did not show any influence. These results indicate that ER-beta in the central nervous system is involved in the anorectic action of estrogen.

Pituitary effects of steroid hormones on secretion of follicle-stimulating hormone and luteinizing hormone

Steroid hormones have a profound influence on the secretion of the gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH). These effects can occur as a result of steroid hormones modifying the secretion of gonadotropin-releasing hormone (GnRH) from the hypothalamus, or a direct effect of steroid hormones on gonadotropin secreting cells in the anterior pituitary gland. With respect to the latter, we have shown that estradiol increases pituitary sensitivity to GnRH by stimulating an increase in expression of the gene encoding the GnRH receptor. Since an estrogen response element (ERE) has not been identified in the GnRH receptor gene, this effect appears to be mediated by estradiol stimulating production of a yet to be identified factor that in turn enhances expression of the GnRH receptor gene. However, the importance of estradiol for enhancing pituitary sensitivity to GnRH during the periovulatory period is questioned because an increase in mRNA for the GnRH receptor precedes the pre-ovulatory rise in circulating concentrations of estradiol. In fact, it appears that the enhanced pituitary sensitivity during the periovulatory period may occur as a result of a decrease in concentrations of progesterone rather than due to an increase in concentrations of estradiol. Estradiol also is capable of altering secretion of FSH and LH in the absence of GnRH. In a recent study utilizing cultured pituitary cells from anestrous ewes, we demonstrated that estradiol induced a dose-dependent increase in secretion of LH, but resulted in a dose-dependent decrease in the secretion of FSH. We hypothesized that the discordant effects on secretion of LH and FSH might arise from estradiol altering the production of some of the intrapituitary factors involved in synthesis and secretion of FSH. To examine this hypothesis, we measured amounts of mRNA for activin B (a factor known to stimulate synthesis of FSH) and follistatin (an activin-binding protein). We found no change in the mRNA for follistatin after treatment of pituitary cells with estradiol, but noted a decrease in the amount of mRNA for activin B. Thus, the inhibitory effect of estradiol on secretion of FSH appears to be mediated by its ability to suppress the expression of the gene encoding activin.

Methoxyestradiols mediate estradiol-induced antimitogenesis in human aortic SMCs.

Estrogen receptors (ERs) are considered to mediate the ability of 17beta-estradiol (estradiol) to reduce injury-induced proliferation of vascular smooth muscle cells (VSMCs), leading to vascular lesions. However, the finding that estradiol attenuates formation of vascular lesions in response to vascular injury in knockout mice that lack either ER-alpha or ER-beta challenges this concept. Our hypothesis is that the local metabolism of estradiol to methoxyestradiols, metabolites of estradiol with little affinity for ERs, mediates the ER-independent antimitogenic effects of estradiol on VSMCs. In human VSMCs, 2-methoxyestradiol and 2-hydroxyestradiol were more potent than was estradiol in inhibiting DNA synthesis (3[H]-thymidine incorporation), collagen synthesis (3[H]-proline incorporation), cell proliferation (cell number), and cell migration (movement of cells across a polycarbonate membrane). The inhibitory effects of estradiol on VSMCs were enhanced by cytochrome-P450 (CYP450) inducers 3-methylcholanthrene and phenobarbital. Moreover, the inhibitory effects of estradiol were blocked in the presence of the CYP450 inhibitor 1-aminobenzotriazole and the catechol-O-methyltransferase inhibitors quercetin and OR486. Both OR486 and quercetin blocked the conversion of 2-hydroxyestradiol to 2-methoxyestradiol; moreover, they blocked the antimitogenic effects of 2-hydroxyestradiol but not of 2-methoxyestradiol. The ER antagonist ICI182780 blocked the inhibitor effects of estradiol on VSMCs, but only at concentrations (>50 micromol/L) that also inhibit the metabolism of estradiol to hydroxyestradiols (precursors of methoxyestradiols). In conclusion, the inhibitory effects of locally applied estradiol on human VSMCs are mediated via a novel ER-independent mechanism involving estradiol metabolism. These findings imply that vascular estradiol metabolism may be an important determinant of the cardiovascular protective effects of estradiol and that nonfeminizing estradiol metabolites may confer cardiovascular protection regardless of gender.