Mice Lacking Estrogen Receptor Research Articles

Role of estrogen signaling in fibroblastic reticular cellsfor innate and adaptive immune responses in antigen-induced arthritis.

Women are more prone to develop rheumatoid arthritis, with peak incidence occurring around menopause. Estrogen has major effects on the immune system and is protective against arthritis. We have previously shown that treatment with estrogen inhibits inflammation and joint destruction in murine models of arthritis, although the mechanisms involved remain unclear. Fibroblastic reticular cells (FRCs) are specialized stromal cells that generate the three-dimensional structure of lymph nodes (LNs). FRCs are vital for coordinating immune responses from within LNs and are characterized by the expression of the chemokine CCL19, which attracts immune cells. The aim of this study was to determine whether the influence of estrogen on innate and adaptive immune cells in arthritis is mediated by estrogen signaling in FRCs. Conditional knockout mice lacking estrogen receptor α (ERα) in CCL19-expressing cells (Ccl19-CreERαfl/fl) were generated and tested. Ccl19-CreERαfl/fl mice and littermate controls were ovariectomized, treated with vehicle or estradiol and subjected to the 28-day-long antigen-induced arthritis model to enable analyses of differentiated T- and B-cell populations and innate cells in LNs by flow cytometry. The results reveal that while the response to estradiol treatment in numbers of FRCs per LN is significantly reduced in mice lacking ERα in FRCs, estrogen does not inhibit joint inflammation or markedly affect immune responses in this arthritis model. Thus, this study validates the Ccl19-CreERαfl/fl strain for studying estrogen signaling in FRCs within inflammatory diseases, although the chosen arthritis model is deemed unsuitable for addressing this question.

226-LB: Genetic Variation of Human Myokine Signaling Is Dominated by Biologic Sex and Sex Hormones

Skeletal muscle physiology is a critical determinant and therapeutic target for type 2 diabetes. Proteins secreted from skeletal muscle, termed myokines, allow the muscle to communicate with other key metabolic organs and several have been established as causal drivers of processes such as exercise and lipid metabolism. Yet, the genetic architecture, regulation, and functions of myokines, as well as degree of conservation of these communication circuits remains inadequately understood. In this study, we performed the first targeted genetic survey of myokine gene regulation and cross-tissue signaling in 310 humans where sex as a biological variable was emphasized. While expression levels of a majority of myokines and cell proportions within skeletal muscle showed little differences between males and females, nearly all significant cross-tissue enrichments operated in a sex-specific or hormone-dependent fashion. Estrogen signaling specifically in muscle showed striking enrichments with myokines, independent of sex-specific signaling. To define the causal roles of estrogen signaling on myokine gene expression and functions, we generated male and female mice lacking estrogen receptor α (Esr1) specifically in skeletal muscle and integrated global RNA-Sequencing with human data. These analyses highlighted mechanisms of sex-dependent myokine signaling conserved between species, such as myostatin enriched for divergent substrate utilization pathways between sexes, and GPX3 as a male-specific link between glycolytic fiber abundance and hepatic inflammation. Collectively, we provide the first genetic survey of human myokines and highlight sex and estrogen receptor signaling as critical variables when assaying myokine functions and how changes in cell composition impact other metabolic organs. This study is currently available on bioRxiv: https://www.biorxiv.org/content/10.1101/2022.01.20.477045v1 Disclosure C. Van: None. M. Seldin: None.

Estradiol suppresses psoriatic inflammation in mice by regulating neutrophil and macrophage functions

Psoriasis is a common inflammatory skin disease resulting from dysregulation of the IL-23/TH17 immune axis. The prevalence and severity of psoriasis is higher in men than in women, although the underlying reasons for this are unclear. We studied whether estradiol, a female hormone, plays protective roles in imiquimod-induced psoriatic inflammation in mice by regulating neutrophil and macrophage functions. Wild-type mice and conditional knockout mice were ovariectomized, supplemented with placebo or estradiol pellets, and an imiquimod-containing cream applied. Mice without endogenous ovarian hormones exhibited exacerbated psoriatic inflammation including increased production of IL-17A and IL-1β, which was reversed by exogenously added estradiol. The suppressive effect of estradiol on the production of IL-1β and IL-17A was abolished in mice lacking estrogen receptors in neutrophils and macrophages (Esr1f/fEsr2f/fLysM-Cre+ mice). IL-1β, which is required for production of IL-17A in the psoriasis model, was mainly produced by neutrophils and inflammatory macrophages. Estradiol suppressed IL-1β production from neutrophils and macrophages in mice both invivo and invitro and from human neutrophils invitro. Our results suggest a novel mechanism for sex-dependent differences in psoriasis clinical phenotypes that may shed new light on the pathology of psoriasis.

Contribution of estrogen to the pregnancy-induced increase in cardiac automaticity

BackgroundThe heart rate progressively increases throughout pregnancy, reaching a maximum in the third trimester. This elevated heart rate is also present in pregnant mice and is associated with accelerated automaticity, higher density of the pacemaker current If and changes in Ca2+ homeostasis in sinoatrial node (SAN) cells. Strong evidence has also been provided showing that 17β-estradiol (E2) and estrogen receptor α (ERα) regulate heart rate. Accordingly, we sought to determine whether E2 levels found in late pregnancy cause the increased cardiac automaticity associated with pregnancy. Methods and resultsVoltage- and current-clamp experiments were carried out on SAN cells isolated from female mice lacking estrogen receptor alpha (ERKOα) or beta (ERKOβ) receiving chronic E2 treatment mimicking late pregnancy concentrations. E2 treatment significantly increased the action potential rate (284 ± 24 bpm, +E2 354 ± 23 bpm, p = 0.040) and the density of If (+52%) in SAN cells from ERKOβ mice. However, If density remains unchanged in SAN cells from E2-treated ERKOα mice. Additionally, E2 also increased If density (+67%) in nodal-like human-induced pluripotent stem cell-derived cardiomyocytes (N-hiPSC-CM), recapitulating in a human SAN cell model the effect produced in mice. However, the L-type calcium current (ICaL) and Ca2+ transients, examined using N-hiPSC-CM and SAN cells respectively, were not affected by E2, indicating that other mechanisms contribute to changes observed in these parameters during pregnancy. ConclusionThe accelerated SAN automaticity observed in E2-treated ERKOβ mice is explained by an increased If density mediated by ERα, demonstrating that E2 plays a major role in regulating SAN function during pregnancy.

1726-P: Male Mice Lacking Estrogen Receptor Beta Develop Obesity but Are Protected from Diet-Induced Insulin Resistance in Skeletal Muscle

Estrogen has an important role in regulating female metabolism. Whereas the role of estrogen receptor (ER) α on female reproductive system is well defined, the role of ERβ (encoded by Esr2) remains unclear. Here we examined the metabolic effects of high-fat diet (HFD) in male Esr2-/- mice on BALB/c background (n=8∼9). After 6 weeks of HFD, Esr2-/- mice became more obese than wild type (WT) mice (Figure 1; *P<0.05). Surprisingly, metabolic cage study showed significantly increased energy expenditure in Esr2-/- mice, likely resulting from skeletal muscle as lean mass tended to be higher in these mice (Figure 2). We performed a hyperinsulinemic-euglycemic clamp to measure insulin sensitivity in awake mice. Despite being more obese, HFD-fed Esr2-/- mice tended to show increased insulin sensitivity with 20% increases in whole body glucose turnover and glycolysis (Figure 3). This was mostly due to a significant increase (52%) in skeletal muscle glucose uptake in HFD-fed Esr2-/- mice (Figure 4). Interestingly, glucose uptake into brown fat tended to be reduced by 50% in HFD-fed Esr2-/- mice. In conclusion, these results indicate that deletion of Esr2 in male mice increases diet-induced obesity, but selectively prevents muscle insulin resistance. Our findings implicate a potential therapeutic role of estrogen signaling to treat insulin resistance. Disclosure R.H. Friedline: None. H. Noh: None. S. Suk: None. C. Goral: None. K.A. Dunphy: None. A.L. Roberts: None. D.A. Tran: None. L.A. Tauer: None. X. Hu: None. L.H. Kim: None. A.M. Kim: None. D. Jerry: None. J.K. Kim: None. Funding National Institutes of Health (5U2CDK093000)

Oviductal Retention of Embryos in Female Mice Lacking Estrogen Receptor α in the Isthmus and the Uterus.

Estrogen receptor α (ESR1; encoded by Esr1) is a crucial nuclear transcription factor for female reproduction and is expressed throughout the female reproductive tract. To assess the function of ESR1 in reproductive tissues without confounding effects from a potential developmental defect arising from global deletion of ESR1, we generated a mouse model in which Esr1 was specifically ablated during postnatal development. To accomplish this, a progesterone receptor Cre line (PgrCre) was bred with Esr1f/f mice to create conditional knockout of Esr1 in reproductive tissues (called PgrCreEsr1KO mice) beginning around 6 days after birth. In the PgrCreEsr1KO oviduct, ESR1 was most efficiently ablated in the isthmic region. We found that at 3.5 days post coitus (dpc), embryos were retrieved from the uterus in control littermates while all embryos were retained in the PgrCreEsr1KO oviduct. Additionally, serum progesterone (P4) levels were significantly lower in PgrCreEsr1KO compared to controls at 3.5 dpc. This finding suggests that expression of ESR1 in the isthmus and normal P4 levels allow for successful embryo transport from the oviduct to the uterus. Therefore, alterations in oviductal isthmus ESR1 signaling and circulating P4 levels could be related to female infertility conditions such as tubal pregnancy.

Abstract P148: Transcriptomic and Proteomic Analysis of CD4 + T Cells to Identify Sex Differences in Angiotensin II Signaling Pathways

T cells are involved in hypertension pathogenesis in both males and postmenopausal females while premenopausal females are resistant to T cell-mediated Ang II-induced hypertension. The goals of this study were (1) to identify T cell specific proteomic pathways associated with postmenopausal susceptibility to hypertension (2) to identify T cell specific transcriptomic changes associated with premenopausal protection from hypertension. Proteomic analysis was performed on splenic CD4 + T cells isolated from premenopausal and postmenopausal females (VCD, 160 mg/kg/day i.p. 20d) following Ang II infusion (800 ng/kg/min 14d). 384 proteins from CD4 + T cells were identified as differentially expressed following Ang II infusion in premenopausal females. 285 proteins from CD4 + T cells were identified as differentially expressed between premenopausal and postmenopausal females following Ang II infusion. Gene ontology (GO) analysis of pre vs. postmenopausal proteins identified enriched pathways associated with RNA binding, chaperone activity and cellular stress responses. Transcriptomic changes were analyzed, via RNAseq, on isolated splenic CD4 + T cells from premenopausal females, with and without Ang II infusion. Thirty-four genes were identified as differentially expressed in CD4 + T cells following Ang II infusion. GO analysis of Ang II upregulated genes revealed an enrichment of five distinct molecular functions, including antioxidant activity. In a subsequent study to validate the RNAseq, we confirmed that Ang II increased CD4 + T cell mRNA expression of calprotectin (S100a8/S100a9), a calcium and zinc binding protein complex that contributes to antioxidant defense (S100a8: Con 1.0 ± 0.4 vs Ang II 5.0 ± 0.8*; S100a9: Con 1.0 ± 0.4 vs Ang II 6.0 ± 0.8*, *P<0.05 vs Con). Furthermore, we determined that Ang II did not increase calprotectin expression in mice lacking estrogen receptor α (ERKO) (S100a8: ERKO 0.1 ± 0.4 vs ERKO/Ang II 0.3 ± 0.7; S100a9: ERKO 0.2 ± 0.5 vs ERKO/Ang II 0.3 ± 0.6). The current studies demonstrate a role for estrogen in Ang II-induced T cell gene expression and signal transduction, and begin to elucidate the molecular mechanisms of female protection from T cell-mediated hypertension.

Impairments in the reproductive axis of female mice lacking estrogen receptor β in GnRH neurons.

The effect of estrogen on the differentiation and maintenance of reproductive tissues is mediated by two nuclear estrogen receptors (ERs), ERα, and ERβ. Lack of functional ERα and ERβ genes in vivo significantly affects reproductive function; however, the target tissues and signaling pathways in the hypothalamus are not clearly defined. Here, we describe the generation and reproductive characterization of a complete-ERβ KO (CERβKO) and a GnRH neuron-specific ERβKO (GERβKO) mouse models. Both ERβKO mouse models displayed a delay in vaginal opening and first estrus. Hypothalamic gonadotropin-releasing hormone (GnRH) mRNA expression levels in both ERβKO mice were similar to control mice; however female CERβKO and GERβKO mice had lower basal and surge serum gonadotropin levels. Although a GnRH stimulation test in both female ERβKO models showed preserved gonadotropic function in the same animals, a kisspeptin stimulation test revealed an attenuated response by GnRH neurons, suggesting a role for ERβ in normal GnRH neuron function. No alteration in estrogen-negative feedback was observed in either ERβKO mouse models after ovariectomy and estrogen replacement. Further, abnormal development of ovarian follicles with low serum estradiol levels and impairment of fertility were observed in both ERβKO mouse models. In male ERβKO mice, no differences in the timing of pubertal onset or serum luteinizing hormone and follicle-stimulating hormone levels were observed as compared with controls. Taken together, these data provide in vivo evidence for a role of ERβ in GnRH neurons in modulating puberty and reproduction, specifically through kisspeptin responsiveness in the female hypothalamic-pituitary-gonadal axis.

Resveratrol Improved Flow-Mediated Outward Arterial Remodeling in Ovariectomized Rats with Hypertrophic Effect at High Dose.

ObjectivesChronic increases in blood flow in resistance arteries induce outward remodeling associated with increased wall thickness and endothelium-mediated dilatation. This remodeling is essential for collateral arteries growth following occlusion of a large artery. As estrogens have a major role in this remodeling, we hypothesized that resveratrol, described as possessing phytoestrogen properties, could improve remodeling in ovariectomized rats.MethodsBlood flow was increased in vivo in mesenteric arteries after ligation of adjacent arteries in 3-month old ovariectomized rats treated with resveratrol (5 or 37.5 mg/kg per day: RESV5 or RESV37.5) or vehicle. After 2 weeks arterial structure and function were measured in vitro in high flow (HF) and normal flow (NF) arteries isolated from each rat.ResultsArterial diameter was greater in HF than in NF arteries in ovariectomized rats treated with RESV5 or RESV37.5, not in vehicle-treated rats. In mice lacking estrogen receptor alpha diameter was equivalent in HF and NF arteries whereas in mice treated with RESV5 diameter was greater in HF than in NF vessels. A compensatory increase in wall thickness and a greater phenylephrine-mediated contraction were observed in HF arteries. This was more pronounced in HF arteries from RESV37.5-treated rats. ERK1/2 phosphorylation, involved in hypertrophy and contraction, were higher in RESV37.5-treated rats than in RESV5- and vehicle-treated rats. Endothelium-dependent relaxation was greater in HF than in NF arteries in RESV5-treated rats only. In HF arteries from RESV37.5-treated rats relaxation was increased by superoxide reduction and markers of oxidative stress (p67phox, GP91phox) were higher than in the 2 other groups.ConclusionResveratrol improved flow-mediated outward remodeling in ovariectomized rats thus providing a potential therapeutic tool in menopause-associated ischemic disorders. This effect seems independent of the estrogen receptor alpha. Nevertheless, caution should be taken with high doses inducing excessive contractility and hypertrophy in association with oxidative stress in HF arteries.

Oviductal estrogen receptor α signaling prevents protease-mediated embryo death

Development of uterine endometrial receptivity for implantation is orchestrated by cyclic steroid hormone-mediated signals. It is unknown if these signals are necessary for oviduct function in supporting fertilization and preimplantation development. Here we show that conditional knockout (cKO) mice lacking estrogen receptor α (ERα) in oviduct and uterine epithelial cells have impaired fertilization due to a dramatic reduction in sperm migration. In addition, all successfully fertilized eggs die before the 2-cell stage due to persistence of secreted innate immune mediators including proteases. Elevated protease activity in cKO oviducts causes premature degradation of the zona pellucida and embryo lysis, and wild-type embryos transferred into cKO oviducts fail to develop normally unless rescued by concomitant transfer of protease inhibitors. Thus, suppression of oviductal protease activity mediated by estrogen-epithelial ERα signaling is required for fertilization and preimplantation embryo development. These findings have implications for human infertility and post-coital contraception.

The Naturally Occurring Luteinizing Hormone Surge Is Diminished in Mice Lacking Estrogen Receptor Beta in the Ovary1

Female ESR2-null mice (betaERKO) display defects in ovarian function and are subfertile. Follicular maturation is impaired and explains smaller litters, but betaERKO also produce fewer litters, which may be partially due to inadequate ovulatory signals. To test this, the amplitude and timing of the naturally occurring luteinizing hormone (LH) surge was measured in individual intact betaERKO and wild-type (WT) mice. Vaginal cytology was evaluated daily, and blood samples were taken from mice in proestrus. The amplitude of the LH surge was severely blunted in betaERKO mice compared to WT, but pituitary LH levels revealed no differences. The betaERKO mice did not produce a preovulatory estradiol surge. To determine if the smaller LH surges and the reduced number of litters in betaERKO were due to the lack of ESR2 in the hypothalamic-pituitary axis or due to the absence of ESR2 in the ovary, ovaries were transplanted from WT into betaERKO mice and vice versa. The size of the LH surge was reduced only in mice lacking ESR2 within the ovary, and these mice had fewer litters. Fertility and size of the LH surge were rescued in betaERKO mice receiving a WT ovary. These data provide the first experimental evidence that the LH surge is impaired in betaERKO females and may contribute to their reduced fertility. ESR2 is not necessary within the pituitary and hypothalamus for the generation of a normal LH surge and for normal fertility, but ESR2 is essential within the ovary to provide proper signals.

VEGF signaling is disrupted in the hearts of mice lacking estrogen receptor alpha

Estrogen has widely been credited for cardioprotection in women. However, the exact mechanisms that underlie these beneficial estrogenic effects are not completely understood. Here, we sought to: 1) elucidate estrogen's influence on levels of vascular endothelial growth factor (VEGF), a key regulator of cardiovascular processes, and components of its basic signaling machinery (VEGF receptors, Akt, and eNOS) in the heart, and 2) delineate the specific estrogen receptor signaling pathway that mediates its beneficial effects using mice lacking either estrogen receptor alpha or estrogen receptor beta. We analyzed pattern of VEGF signaling and the associated coronary capillary density in the hearts of wild-type (WT), estrogen receptor alpha knockout (ERα-KO), and estrogen receptor beta knockout (ERβ-KO) female mice. Deletion of estrogen receptor alpha causes a marked decrease in coronary capillary density compared to wild-type (WT) mice, while that of estrogen receptor beta had a minimal effect. Consistent with reduced coronary capillary density, cardiac expression levels of VEGF and its signaling molecules (two receptors, phosphorylated Akt, and eNOS) in ERα-KO mice were reduced to half of WT, in contrast to ERβ-KO mice that only showed a slight decrease. Moreover, activity of eNOS was greatly lowered in ERα-KO mice. These data suggest that estrogen acts largely via estrogen receptor alpha to regulate VEGF transcription and possibly components of its basic signaling and ultimately, the development of coronary microvasculature in the heart. This molecular and histological data, in part, sheds some insights into potential mechanisms that may likely underlie estrogen's cardioprotective effects.

Estrogen signaling is not required for prostatic bud patterning or for its disruption by 2,3,7,8-tetrachlorodibenzo- p-dioxin

Estrogens play an important role in prostatic development, health, and disease. While estrogen signaling is essential for normal postnatal prostate development, little is known about its prenatal role in control animals. We tested the hypothesis that estrogen signaling is needed for normal male prostatic bud patterning. Budding patterns were examined by scanning electron microscopy of urogenital sinus epithelium from wild-type mice, mice lacking estrogen receptor (ER)α, ERβ, or both, and wild-type mice exposed to the antiestrogen ICI 182,780. Budding phenotypes did not detectably differ among any of these groups, strongly suggesting that estrogen signaling is not needed to establish the prototypical prostatic budding pattern seen in control males. This finding contributes to our understanding of the effects of low-level estrogen exposure on early prostate development. In utero exposure to 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) can greatly alter the pattern in which prostatic buds form and reduce their number. For several reasons, including a prior observation that inhibitory effects of TCDD on prostatic budding in rats depend heavily on the sex of adjacent fetuses, we tested the hypothesis that estrogen signaling is needed for TCDD to disrupt prostatic budding. However, budding did not detectably differ among wild-type mice, or mice lacking ERα, ERβ, or both, that were exposed prenatally to TCDD (5 μg/kg on embryonic day 13.5). Nor did ICI 182,780 detectably affect the response to TCDD. These results strongly suggest that estrogen signaling is not needed for TCDD to inhibit prostatic epithelial budding.

Impaired Estrogen Feedback and Infertility in Female Mice with Pituitary-Specific Deletion of Estrogen Receptor Alpha (ESR1)1

Mice lacking estrogen receptor alpha in the pituitary gonadotroph (PitEsr1KO) were generated to determine the physiologic role of pituitary estrogen signaling in the reproductive axis. PitEsr1KO female mice are subfertile or infertile and have elevated levels of serum luteinizing hormone (LH) and LH beta subunit gene expression, reflecting a lack of estrogen negative feedback effect on the gonadotroph. While serum LH values are elevated in PitEsr1KO mice, the degree of elevation is much less than that observed in ESR1-null mice, indicating that the hypothalamus must also have an important role in estrogen negative feedback. PitEsr1KO mice also demonstrate a defect in estrogen positive feedback, as surge LH values and estrous cyclicity are absent in these mice. Although sex steroid feedback in the reproductive axis is thought to involve discrete anatomic regions that mediate either a positive or negative estrogen effect, PitEsr1KO mice demonstrate novel evidence that localizes both estrogen positive feedback and estrogen negative feedback to the gonadotroph, which suggests that they may be mechanistically related.

Estrogen Receptor β Selective Ligand 5α-Androstane-3β, 17β-Diol Stimulates Spermatogonial Deoxyribonucleic Acid Synthesis in Rat Seminiferous Epithelium in Vitro

Gonadotropins and testosterone are important regulators of spermatogenesis, even though gonadotropin receptors and the androgen receptor are not expressed by germ cells. However, a functional role for estrogens in connection with male reproduction has been postulated on the basis of the phenotypes of mice lacking estrogen receptor (ER) and cytochrome P-450 aromatase. This has further support by findings of ER expression in the testis, including that of ERbeta in spermatogonia. 5alpha-Androstane-3beta, 17beta-diol (3betaAdiol), a metabolite of testosterone produced via the intermediate potent androgen 5alpha-dihydrotestosterone (DHT), has been reported to selectively bind ERbeta rather than EpsilonRalpha, but not androgen receptor. Here, we have characterized the influence of 17beta-estradiol (E), the major physiological estrogen, 3betaAdiol, and DHT on DNA synthesis in vitro by segments of the seminiferous epithelium at different stages of the seminiferous epithelial cycle in the rat. E and 3betaAdiol exerted similar stimulatory effects on premitotic DNA synthesis in stage I segments, whereas other stages tested (V, VIIa, and XIII-IX) remained unresponsive. In contrast, DHT had no effect on this process. 5-bromo-2'-deoxyuridine labeling of stage I segments revealed a 30-fold higher labeling index in the presence than in the absence of E, and the labeled cells were identified as spermatogonia. Moreover, high levels of 3betaAdiol were found in the testis of intact rats as well as in primary cultures of rat Leydig cells in response to human chorionic gonadotropin. We suggest that 3betaAdiol may serve as a growth factor for germ cells stimulating premitotic DNA synthesis in connection with spermatogenesis via an ERbeta-dependent pathway.

Apoptosis prevents osteoporosis

Grandmothers everywhere know well that estrogen deficits lead to osteoporosis. Now, the molecular basis for this debilitating bone loss is finally identified. Estrogen is needed to kill off bone-destroying osteoclasts, show Takashi Nakamura, Shigeaki Kato (University of Tokyo, Japan), and colleagues. Figure 1 The survival of osteoclasts (top, purple) in mice lacking estrogen receptors (right) leads to loss of bone mass (bottom). The root cause of osteoporosis has been difficult to pin down, in part because bones are not frail in female mice lacking estrogen receptors. These mice make extra androgen, which builds bone in male mice and might compensate for bone loss in the mutant females. To avoid the androgen rise, Kato's group knocked out estrogen receptors only in mature osteoclasts, which accumulate in osteoporotic bones. These female mutants developed rickety bones due to losses within the central bone shafts. The authors then isolated osteoclasts to determine why they are so abundant in diseased bone. Microarray analyses revealed that estrogen induced apoptotic proteins, including Fas ligand, that were not induced in the estrogen-blind osteoclasts. Men who have estrogen receptor mutations develop osteoporosis. But male mice were not affected by the loss of estrogen receptors in osteoclasts. Perhaps the androgen-headed pathway is more dominant in mice than in humans. Currently, potential drugs to treat osteoporosis are screened through mice whose ovaries have been removed. Screens for the induction of Fas ligand in cultures of estrogen-blind osteoclasts should be much simpler. Reference: Nakamura, T., et al. 2007. Cell. 130:811–823. [PubMed]

A Novel Ligand-independent Function of the Estrogen Receptor Is Essential for Osteocyte and Osteoblast Mechanotransduction

Bone senses and adapts to meet mechanical needs by means of an extensive mechanotransduction network comprising osteocytes (former osteoblasts entrapped in mineral) and their cytoplasmic projections through which osteocytes communicate with osteoblasts and osteoclasts on the bone surface. Mechanical stimulation promotes osteocyte (and osteoblast) survival by activating the extracellular signal-regulated kinases, ERKs. Estrogens have similar effects and, intriguingly, the adaptive response of bone to mechanical forces is defective in mice lacking estrogen receptor (ER) alpha or ERbeta. We report that ERKs are not activated by stretching in osteocytic and osteoblastic cells in which both ERalpha and ERbeta have been knocked out or knocked down and this is reversed partially by transfection of either one of the two human ERs and fully by transfection of both receptors. ERK activation in response to stretching is also recovered by transfecting the ligand-binding domain (E) of either receptor or an ERalpha mutant that does not bind estrogens. Furthermore, mechano-responsiveness is restored by transfecting the Ealpha targeted to the plasma membrane, but not to the nucleus, whereas ERalpha mutants with impaired plasma membrane localization or binding to caveolin-1 fail to confer ERK activation in response to stretching. Lastly, the ER antagonist ICI 182,780 abrogates ERK activation and the anti-apoptotic effect of mechanical stimulation. We conclude that in addition to their role as ligand-dependent mediators of the effects of estrogens, the ERs participate in the transduction of mechanical forces into pro-survival signaling in bone cells, albeit in a ligand-independent manner.

Behavioral and metabolic phenotyping of GPR30 and neuronal estrogen receptor- α knockout mice.

We have been testing the hypothesis that estrogen signaling through specific estrogen receptors contributes to the regulation of energy homeostasis and behavior. Mice lacking estrogen receptor alpha (ERα) have previously been reported to have an obese phenotype and attenuated locomotor activity. We have extended these findings to show that estrogen signaling through hypothalamic ERα contributes to the regulation of food intake, body weight, and energy expenditure. To better understand the hypothalamic contribution of estrogen signaling, we first characterized some behavioral aspects of the whole-body ERα knockout mouse, and found that they had decreased homecage locomotor activity and voluntary wheel running activity, increased body weight, and changes in fat distribution to favor development of the metabolic syndrome. They were also resistant to the effects of leptin to regulate food intake and body weight. Recently, we have obtained ERα floxed mice and have bred them with nestin-CRE mice to develop mice lacking estrogen receptors in neurons. We have begun characterization of these mice through multiple metabolic and behavioral tests to test the hypothesis that estrogen signaling through neuronal ERα significantly contributes to the phenotype seen in total body ERα knockout mice. Finally, in addition to nuclear receptors, the actions of estrogen may be mediated through GPR30, a membrane/intracellular receptor. Here, we report on metabolic and behavioral phenotyping assays of GPR30 knockout mice, designed to delineate whether the observed phenotype is mediated through genomic effects or by rapid signaling through membrane receptors.

63: Cystitis and Nociception in Mice Lacking Estrogen Receptors

You have accessJournal of UrologyPodium, Saturday, May 20, 2006, 1:00 - 3:00 pm1 Apr 200663: Cystitis and Nociception in Mice Lacking Estrogen Receptors Zun-Yi Wang, Peiqing Wang, and Dale E. Bjorling Zun-Yi WangZun-Yi Wang More articles by this author , Peiqing WangPeiqing Wang More articles by this author , and Dale E. BjorlingDale E. Bjorling More articles by this author View All Author Informationhttps://doi.org/10.1016/S0022-5347(18)32330-9AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail "63: Cystitis and Nociception in Mice Lacking Estrogen Receptors." The Journal of Urology, 175(4S), pp. 21–22 © 2016 by American Urological AssociationFiguresReferencesRelatedDetails Volume 175Issue 4SApril 2006Page: 21-22 Advertisement Copyright & Permissions© 2016 by American Urological AssociationMetricsAuthor Information Zun-Yi Wang More articles by this author Peiqing Wang More articles by this author Dale E. Bjorling More articles by this author Expand All Advertisement Loading ...

Estrogen up-regulates hepatic expression of suppressors of cytokine signaling-2 and -3 in vivo and in vitro.

Suppressors of cytokine signaling (SOCS) are important negative regulators of cytokine action. We recently reported that estrogen stimulates SOCS-2 expression and inhibits GH signaling in kidney cells. The effects of estrogen on SOCS expression in other tissues are unclear. The aim of this study was to investigate in vivo and in vitro whether estrogen affected SOCS expression in the liver, a major target organ of GH. The in vivo hepatic effects of estrogen on ovariectomized mice lacking estrogen receptor (ER)-alpha, ERbeta, or both and their wild-type littermates were examined by DNA microarray analysis. In vitro, the effects of estrogen on SOCS expression in human hepatoma cells were examined by reverse transcription quantitative PCR. Long-term (3 wk) estrogen treatment induced a 2- to 3-fold increase in hepatic expression of SOCS-2 and -3 in wild-type and ERbeta knockout mice but not in those lacking ERalpha or both ER subtypes. Short-term treatment (at 24 h) increased the mRNA level of SOCS-3 but not SOCS-2. In cultured hepatoma cells, estrogen increased SOCS-2 and -3 mRNA levels by 2-fold in a time- and dose-dependent manner (P < 0.05). Estrogen induced murine SOCS-3 promoter activity by 2-fold (P < 0.05) in constructs containing a region between nucleotides -1862 and -855. Moreover, estrogen and GH had additive effects on the SOCS-3 promoter activity. In summary, estrogen, via ERalpha, up-regulated hepatic expression of SOCS-2 and -3, probably through transcriptional activation. This indicates a novel mechanism of estrogen regulation of cytokine action.