Physiological Doses Of E2 Research Articles

Treatment with a tissue-selective oestrogen complex does not affect disease pathology but reduces pre-BI cells in lupus-prone mice

Objective Systemic lupus erythematosus (SLE or lupus) is an autoimmune disease characterized by B-cell dysfunction, production of autoantibodies, and immune complex formation. Lupus is overrepresented in females, indicating that sex hormones play a role in the pathophysiology. Treatment with a tissue-selective oestrogen complex (TSEC) containing conjugated oestrogens and the selective oestrogen receptor modulator bazedoxifene (BZA) protects against postmenopausal vasomotor symptoms and osteoporosis, but its impact on organ damage in lupus is not fully understood. Method We used ovariectomized MRL/lpr mice, treated with two different physiological doses of 17β-oestradiol-3-benzoate (E2), BZA, or TSEC (E2 plus BZA), to assess early and late B-cell development and to determine histological disease manifestations in the kidneys and salivary glands. Results TSEC treatment reduced the frequency of the pre-BI population in bone marrow to levels equivalent to treatment with physiological doses of E2 alone but did not affect any of the other examined B-cell populations. Our earlier studies indicated that TSEC treatment did not aggravate disease development in ovariectomized MRL/lpr mice, while protecting against trabecular bone loss. Here, we follow up on our previous study and show that neither ovariectomy alone nor TSEC treatment of ovariectomized MRL/lpr mice influenced perivascular lymphocyte infiltration to the kidneys or salivary glands. Conclusion TSEC does not aggravate a mouse model of lupus, when given in doses that protect against postmenopausal lupus-associated bone loss. This indicates that further investigations into TSEC as a treatment for osteoporosis or vasomotor symptoms in postmenopausal women with SLE are warranted.

Abstract 400: Role of the 17beta-Estradiol/beta-Catenin Axis in Heart Failure

The role of β-catenin and the actions of 17β-estradiol (E2) in cardiovascular (patho)physiology remain unclear. In the diseased heart, E2 is expected to be cardioprotective. However, in the absence of prior disease, complications in pregnancy or transgender individuals indicate that E2 might lead to cardiovascular disease. We aimed at the characterization of the effects of the E2/β-catenin axis in the healthy C57Bl/6N heart. Two-month-old female C57Bl/6N wild-type (WT) and cardiac-specific β-catenin-deleted (β-cat Δex2-6 ) mice were ovariectomized and randomized to an E2-containing or soy-free (control, CON) diet ( n = 7-13/group). The 3-month physiological dose of E2 led to a significant repression of nuclear β-catenin levels vs. CON ( P < 0.001) in WT mice. This was underlain by a significant decrease in the heart weight to tibia length ratio and cardiomyocyte cross-sectional area (E2 vs. CON WT mice P < 0.001). Echocardiography revealed a significant decrease in septum width ( P < 0.001), posterior wall thickness ( P < 0.01) and fractional shortening ( P < 0.05) in E2 vs. CON WT mice. Deletion of β-catenin abrogated these E2-mediated deleterious effects on cardiac structure and function. Mechanistically, we found a significant increase in the levels of the ubiquitin ligase and key regulator of proteasome-dependent protein degradation muscle-specific RING finger protein 1 (MuRF1) in E2 vs. CON WT mice ( P < 0.05), while there was no effect in β-cat Δex2-6 mice. Although we also hypothesized increased autophagic activity, we found no effect on the autophagy-related protein LC3 in WT or β-cat Δex2-6 mice. In a translational approach, we profiled the left ventricular transcriptome of female patients ( n = 5) with end-stage non-ischemic dilated cardiomyopathy (DCM) vs. healthy controls ( n = 8). We found a significant induction in the expression of the genes coding for SFRP5 , a negative regulator of Wnt signaling, DACT2 , an antagonist of β-catenin, and APC2 , which is required for targeted degradation of β-catenin ( P < 0.01), in DCM samples. Our mouse data indicate that repression but not deletion of β-catenin leads to heart failure and our human data suggest that targeting Wnt/β-catenin signaling in heart failure may be of therapeutic value.

Abstract 36: Administration of 17β-Estradiol in C57Bl/6N Female Mice Leads to Cardiac Atrophy and Dysfunction via a β-Catenin Mechanism

The steroid hormone 17β-estradiol (E2) regulates several biological processes. In contrast to its anti-hypertrophic effects under pressure overload, we recently found that E2 induced physiological hypertrophic growth in healthy C57Bl/6J mice but not C57Bl/6N mice. Here, we aimed at the characterization of the effects of E2 in C57Bl/6N mice and tested the hypothesis that β-catenin mediates these E2 effects. Following ovariectomy, 2-month-old C57Bl/6N wild-type and cardiac-specific β-catenin-deleted (β-cat Δex2-6 ) mice were randomized to an E2-containing or soy-free (control, CON) diet ( n = 7-13/group). Cardiac function was examined by echocardiography following established procedures. The 3-month physiological dose of E2 led to a higher relative uterus weight compared with CON ( P < 0.001) in both WT and β-cat Δex2-6 mice. The relative heart weight was significantly reduced by E2 compared with CON in WT mice ( P < 0.001), while there was no significant effect in β-cat Δex2-6 mice. Cardiomyocyte cross-sectional area was also significantly decreased by E2 ( n = 5-7/group; P < 0.001) compared with CON in WT mice, while there was no significant effect in β-cat Δex2-6 mice. Echocardiography revealed a significant decrease in septum width ( P < 0.001) and posterior wall thickness ( P < 0.01) in E2 treated WT mice compared with CON, while there was no significant effect in β-cat Δex2-6 mice ( n = 8/group). These E2-induced structural changes in WT mice were accompanied by a significant decrease in cardiac function, namely a 23% decrease in fractional shortening compared with CON ( P < 0.05), while there was no significant effect in β-cat Δex2-6 mice. Immunoblotting revealed a significant increase in the levels of the ubiquitin ligase and key regulator of proteasome-dependent protein degradation muscle-specific RING finger protein 1 (MuRF1) by E2 compared with CON in WT mice ( P < 0.05), while there was no significant effect in β-cat Δex2-6 mice. Although we hypothesized increased autophagic activity, we found no effect on the autophagy-related protein LC3 in WT or β-cat Δex2-6 mice. In conclusion, our surprising findings show that E2 leads to cardiac atrophy and dysfunction in C57Bl/6N mice via a β-catenin mechanism seemingly in an autophagy-independent manner.

Time course of cytokine upregulation in the lacrimal gland and presence of autoantibodies in a predisposed mouse model of Sjögren's Syndrome: The influence of sex hormones and genetic background

Sjögren's Syndrome (SS) is a chronic, inflammatory autoimmune disease characterized by lacrimal gland lymphocytic infiltration and epithelial cell death, as well as by the presence of serum autoantibodies. Although the symptoms of this syndrome are well characterized, patients are not diagnosed until 5–10 years into disease progression; furthermore, the early series of events leading to the initiation of SS are not well understood. In order to better understand the early events of the disease, we have been using ovariectomized (OVX) NOD.B10.H2b mice as a genetically predisposed model of SS. Previously, we have shown that removal of ovarian hormones through ovariectomy accelerated the symptoms of this disease, and in early events of SS in the lacrimal glands, lymphocytic infiltration preceded acinar cell apoptosis. To further elucidate the earlier events of this disease in the SS animal model, we investigated the expression and concentration of pro-inflammatory cytokines in the lacrimal glands as well as the presence of autoantibodies in both lacrimal glands and serum. Six weeks old NOD.B10.H2b and C57BL/10 control mice were either sham-operated, OVX, OVX and treated with 17β-estradiol (E2), or OVX and treated with dihydrotestosterone (DHT). Lacrimal glands were collected at 3, 7, 21, and 30 days after surgery and analyzed for cytokines IL-1β, TNF-α, IFN-γ, IL-10, and IL-4 gene expression by using quantitative RT-PCR and for cytokine levels using ELISA. Furthermore, anti-Ro/SSA and anti-La/SSB autoantibodies were measured in the serum and lacrimal glands supernatants using ELISA. The results of this study showed that OVX caused a significant increase in the expression and levels of the cytokines IL-1β, TNF-α, and IL-4 in the lacrimal glands of the NOD.B10.H2b mice starting at 3 days after OVX, while a significant increase of IL-10 gene expression and levels was observed only at later experimental time points. A small but significant increase in the expression of IL-1β and IL-4 was observed only at later experimental time points in the lacrimal glands of OVX C57BL/10 mice, while no significant changes in the expression of TNF-α and IL-10 were seen at any experimental times in this group. No significant differences were observed in the levels of the cytokines IL-1β, TNF-α, IL-4, and IL-10 in the lacrimal glands of the OVX C57BL/10 mice at any of the experimental times studied compared to the sham-operated group. IFN-γ was not detected in either mouse strains at the level of mRNA and protein. OVX in the NOD.B10.H2b mice also caused an increase in the levels of anti-Ro/SSA autoantibodies in the serum only, while no anti-La/SSB autoantibodies were found in the serum or lacrimal gland supernatants. Physiological doses of E2 or DHT at time of OVX prevented the upregulation of cytokines and the presence of anti-Ro/SSA autoantibodies in these animals. These results showed that a decrease in the concentrations of ovarian hormones in the genetically predisposed mice accelerated the onset of the disease by upregulating various pro-inflammatory cytokines at different time points and promoting the formation of anti-Ro/SSA serum autoantibodies, creating an environment favorable for the initiation of SS.

AB0187 Estrogen-Induction of STAT1 and STAT4 Expression by Estrogen Receptor Alpha Suggests a Novel IFN-Alpha-Independent Mechanism of Sex-Bias in Systemic Lupus Erythematosus Pathogenesis

BackgroundSystemic Lupus Erythematosus (SLE) is a devastating multi-organ autoimmune disease displaying an overwhelming female predilection. Estrogen (E2) influences gene expression by binding to estrogen receptors (ERα and ERβ) and has...

Abstract 055: β-Catenin Mediates an Interplay between Genetic Background and Growth-Regulating Effects of Estrogen in the Healthy Heart

In diseased hearts, estrogen (E2) has been shown to exert anti-hypertrophic actions. Little is known about the role of E2 in the healthy heart. Our initial aim was to characterize structurally and molecularly the effects of E2 in the healthy mouse heart. Two-month-old female C57Bl/6J mice were ovariectomized and randomized to an E2-containing or soy-free (control, CON) diet ( n = 17-18/group). The three-month physiological dose of E2 led to a higher relative heart weight compared with CON ( P < 0.001). We also confirmed increased cardiomyocyte cross-sectional area by E2 ( P < 0.01). No activation of the fetal gene program and no fibrosis were observed. Transcriptome analysis revealed induction of growth-related pathways by E2, such as the Wnt signaling pathway ( n = 5/group; adjusted P < 0.05). To further confirm activation of Wnt/β-catenin signaling, we verified increased nuclear β-catenin protein levels by E2 compared with CON ( P < 0.01) and hypothesized that β-catenin mediates the actions of E2. Cardiac deletion of β-catenin blunted the E2 effects on cardiac growth ( n = 13/group). Surprisingly, in wild-type littermates with the background C57Bl/6N, E2 decreased the relative heart weight and cardiomyocyte cross-sectional area compared with CON ( n = 7-11/group; P < 0.001). This was underlain by decreased nuclear β-catenin protein levels by E2 compared with CON ( P < 0.001). Furthermore, E2 increased glycogen synthase kinase 3β (GSK3β) phosphorylation and the endosomal/autophagosomal markers Rab5, Rab7 and LC3-II in C57Bl/6J but not C57Bl/6N mice. Assessing a polymorphism linked to Snap29 , we confirmed higher Snap29 protein levels in C57Bl/6J than C57Bl/6N mice ( P < 0.01). This could lead to distinct regulation of endosomes and could be the potential cause of the strain difference. In conclusion, E2 regulates cardiac growth through β-catenin in a strain-specific manner. Collectively, we identified a molecular mechanism that demonstrates a divergent response of mouse sub-strains to E2.

A calcium binding protein, Calbindin-D9k, is mainly regulated by estrogen in the pituitary gland of rats during estrous cycle

As a member of family of cytosolic calcium binding proteins, Calbindin-D9k (CaBP-9k) is expressed in female reproductive system and regulated by steroid hormones, estrogen (E2) and progesterone (P4), but its expression and role in pituitary gland have not been elucidated yet. Thus, in this study, we elucidated the expression of CaBP-9k mRNA and protein in pituitary gland of rats. During estrous cycle of rats, pituitary CaBP-9k level fluctuated, and its mRNA was highly elevated during an E2-dominant stage (proestrus and estrus), whereas its level disappeared at a P4-dominant stage (metestrus and diestrus). In parallel with CaBP-9k mRNA, an increased level of CaBP-9k protein was observed during proestrus and estrus, suggesting that pituitary CaBP-9k may be up-regulated by E2. In addition, spatial CaBP-9k expression was attested by immunohistochemistry. Pituitary CaBP-9k protein was localized in the cytoplasm of a specific cell type in the anterior lobe, and the positive cells were abundant at proestrus and estrus. The CaBP-9k-positive cells were mainly localized in the acidopils producing growth hormones and prolactin. To verify hormonal regulation of pituitary CaBP-9k in this tissue, immature rats were treated with a physiological dose of E2 in the absence or presence P4 for 3 days. In a time-dependent experiment, pituitary CaBP-9k protein was induced at 48 h after the final E2 injection. A significant increase in CaBP-9k protein was caused by E2, whereas P4 antagonized E2-stimulated CaBP-9k expression as similarly observed in the uterus. Taken together, these results indicated for the first time that pituitary CaBP-9k expression is regulated during estrous cycle, and its expression is mainly controlled by E2 and antagonized by P4, suggesting that pituitary CaBP-9k in female rats may involve in the central function of the reproduction system.

Disruption of estrogen receptor beta gene impairs spatial learning in female mice.

Here we provide the first evidence, to our knowledge, that estradiol (E(2)) affects learning and memory via the newly discovered estrogen receptor beta (ERbeta). In this study, ERbeta knockout (ERbetaKO) and wild-type littermates were tested for spatial learning in the Morris water maze after ovariectomy, appropriate control treatment, or one of two physiological doses of E(2). Regardless of treatment, all wild-type females displayed significant learning. However, ERbetaKOs given the low dose of E(2) were delayed in learning acquisition, and ERbetaKOs administered the higher dose of E(2) failed to learn the task. These data show that ERbeta is required for optimal spatial learning and may have implications for hormone replacement therapy in women.

Biphasic effects of the natural estrogen 17beta-estradiol on hepatic cholesterol metabolism in intact female rats.

The protective influence of estrogens in cardiovascular disease is believed to be partly due to beneficial effects on cholesterol metabolism. Much of the experimental data are based on models in which synthetic estrogens have been used in pharmacological doses, and therefore, the physiological role of estrogens in cholesterol metabolism is uncertain. To evaluate this important issue, we performed experiments in intact female rats with use of the natural estrogen 17beta-estradiol (E2) administered either subcutaneously or orally. After physiological doses of E2 (< or =0.04 mg. kg(-1). d(-1)) were administered, plasma levels of high density lipoprotein (HDL) cholesterol and apolipoprotein (apo) A-I were increased. In the liver, 3-hydroxy-3-methylglutaryl coenzyme A reductase and cholesterol 7alpha-hydroxylase activities were increased, as well as cholesterol 7alpha-hydroxylase mRNA levels. These effects were abolished during treatment with higher doses of E2, whereas apo A-I mRNA increased in a dose-dependent way. After treatment with pharmacological doses of E2 (> or =0.2 mg. kg(-1). d(-1)), the number of hepatic low density lipoprotein receptors increased and plasma cholesterol was reduced. These effects were similar after both oral and subcutaneous administration of E2. Our results show that the responses to E2 are biphasic: plasma HDL, apo A-I, and hepatic enzyme activities governing bile acid and cholesterol synthesis increased only at physiological doses of E2. At pharmacological doses of E2, hepatic low density lipoprotein receptors are stimulated and plasma cholesterol is reduced. Therefore, under physiological conditions, E2 exerts its major effects on hepatic cholesterol metabolism through mechanisms other than stimulation of low density lipoprotein receptor expression.

Alterations in mammary gland development following neonatal exposure to estradiol, transforming growth factor α, and estrogen receptor antagonist ICI 182,780

High fetal/early postnatal levels of estrogen increase breast cancer risk, but the mechanisms remain unknown. Growth factors, such as transforming growth factor α (TGFα), may participate as secondary modifiers in this process. We characterized a modulatory role of early postnatal exposure to 17β-estradiol (E2) on the developing mammary gland morphology by treating intact female CD-1 mice with physiological doses of E2 (2–4 μg), human recombinant TGFα (4 μg), or an estrogen receptor (ER) antagonist ICI 182,780 (20 μg) during postnatal days 1–3. Early postnatal exposure of E2 stimulated mammary ductal growth by days 25 and 35, but by day 50 this was inhibited. The level of differentiation from terminal end buds (TEBs) to the lobulo-alveolar units (LAUs) also was reduced by day 50. The number of TEBs was increased throughout most of the development in the female mice exposed to E2 during early life. An exposure to TGFα or ICI 182,780 between postnatal days 1 and 3 stimulated ductal growth, formation of TEBs, and the differentiation of mammary epithelial structures. ICI 182,80 treatment also caused hyperplastic lobular-like structures in 54-day-old females. Thus, neonatal exposure to TGFα and ICI 182,780 induced both similar (increase in TEBs) and different (increase/decrease in lobulo-alveolar differentiation) developmental changes in the mouse mammary gland, when compared with an exposure to E2. A unique feature of the postnatal E2 treatment was that it inhibited ductal migration by days 50–54. Our data suggest than an exposure to E2 on postnatal days 1–3, possibly combined with secondary epigenetic alterations, leads to various changes within the developing mammary tree. These changes may be potential prerequisites for mammary tumorigenesis. J. Cell. Physiol. 170:279–289, 1997. © 1997 Wiley-Liss, Inc.

Alterations in mammary gland development following neonatal exposure to estradiol, transforming growth factor alpha, and estrogen receptor antagonist ICI 182,780.

High fetal/early postnatal levels of estrogen increase breast cancer risk, but the mechanisms remain unknown. Growth factors, such as transforming growth factor alpha (TGF alpha), may participate as secondary modifiers in this process. We characterized a modulatory role of early postnatal exposure to 17 beta-estradiol (E2) on the developing mammary gland morphology by treating intact female CD-1 mice with physiological doses of E2 (2-4 micrograms), human recombinant TGF alpha (4 micrograms), or an estrogen receptor (ER) antagonist ICI 182,780 (20 micrograms) during postnatal days 1-3. Early postnatal exposure of E2 stimulated mammary ductal growth by days 25 and 35, but by day 50 this was inhibited. The level of differentiation from terminal end buds (TEBs) to the lobulo-alveolar units (LAUs) also was reduced by day 50. The number of TEBs was increased throughout most of the development in the female mice exposed to E2 during early life. An exposure to TGF alpha or ICI 182,780 between postnatal days 1 and 3 stimulated ductal growth, formation of TEBs, and the differentiation of mammary epithelial structures. ICI 182,80 treatment also caused hyperplastic lobular-like structures in 54-day-old females. Thus, neonatal exposure to TGF alpha and ICI 182,780 induced both similar (increase in TEBs) and different (increase/decrease in lobulo-alveolar differentiation) developmental changes in the mouse mammary gland, when compared with an exposure to E2. A unique feature of the postnatal E2 treatment was that it inhibited ductal migration by days 50-54. Our data suggest than an exposure to E2 on postnatal days 1-3, possibly combined with secondary epigenetic alterations, leads to various changes within the developing mammary tree. These changes may be potential prerequisites for mammary tumorigenesis.

Estrogen induced suppression of collagen arthritis V: Physiological level of estrogen in [formula omitted] mice is therapeutic on established arthritis, suppresses anti-type II collagen T-cell dependent immunity and stimulates polyclonal B-cell activity

Immunization of castrated female DBA 1 mice with rat type II collagen (CII) induces severe polyarthritis with an onset 3–5 weeks after immunization and with 80–100% incidence. Estrogen treatment, inducing physiological 17β-estradiol (E2) levels, during a limited period before and after the immunization, or during another period before the expected onset of arthritis, delayed the arthritic onset by approximately 10 days but did not affect the incidence of severity of arthritis. Treatment with physiological doses of E2 after onset of arthritis decreased severity and duration of disease. The T-cell dependent anti-CII autoantibody response was suppressed if the E2 treatment was given immediately before and after CII immunization and was not significantly affected if E2 treatment was given after CII immunization. Neither the total anti-CII Ig levels nor the anti-CII IgG2a levels correlated with development of arthritis. We also titrated the serum levels of estrogen and recorded the vaginal smear response after injections of various doses of E2. This enabled us to work in a physiological range of estrogen levels, spanning the levels found at the end of pregnancy and those found during the normal estrous cycle. These levels were found to suppress antigen-specific T-cell functions but enhance certain B-cell activities since the delayed type hypersensitivity (DTH) reaction against CII was suppressed while the total number of splenic Ig-secreting cells increased. These findings suggest that estrogen in physiological doses is therapeutic for the development of collagen-induced arthritis and that estrogen exerts dualistic effects on the immune system by suppressing T-cell functions and stimulating certain B-cell activities. The suppressive effect on arthritis could not be explained by suppression of anti-CII autoantibody response and must therefore depend on other T-cell-mediated functions.

Differential Feedback Regulation by 17β-Estradiol of LH Production and LHRH-Responsiveness in Adenohypophyseal Cell Cultures

To evaluate the direct effects of 17β-estradiol (E2) on pituitary LH release, cellular LH content, and LHRH-gesponsive-ness, physiological doses of E2 (10−10, 10−9, or 10−8M) were tested in pituitary monolayer cultures of female rats. The effects of E2 appeared to be dose-related. After a4 h incubation, basal LH release was significantly stimulated by lO−10M E2 (176% of control level) whereas 10−10M or 10−8M had no effect. The cellular LH content was significantly stimulated by 10−8M E2 (127% of control level) but not by the lower doses of E2. Both 10−8M and 10−9m E2 but not 10−10M E2 stimulated LH production (sum of LH contents in medium and cells). When the cultures were incubated with 10−8M LHRH, cellular LH content was depressed (76% of control level) by 10−8M E2 coincubation, whereas LH release was not altered. To further explore the interaction between E2 and LHRH, extended preincubation times with 10−8M E2 were used to test its effect on the subsequent LHRH-responsiveness. After a lag period, prein...