Estrogen Receptor (ER)-β Reduces ERα-Regulated Gene Transcription, Supporting a “Ying Yang” Relationship between ERα and ERβ in Mice

Abstract Estrogen is of importance for the regulation of adult bone metabolism. The aim of the present study was to determine the role of estrogen receptor-β (ERβ) in vivo on global estrogen-regulated transcriptional activity in bone. The effect of estrogen in bone of ovariectomized mice was determined using microarray analysis including 9400 genes. Most of the genes (95% = 240 genes) that were increased by estrogen in wild-type (WT) mice were also increased by estrogen in ERβ-inactivated mice. Interestingly, the average stimulatory effect of estrogen on the mRNA levels of these genes was 85% higher in ERβ-inactivated than in WT mice, demonstrating that ERβ reduces estrogen receptor-α (ERα)-regulated gene transcription in bone. The average stimulatory effect of estrogen on estrogen-regulated bone genes in ERα-inactivated mice was intermediate between that seen in WT and ERαβ double-inactivated mice. Thus, ERβ inhibits ERα-mediated gene transcription in the presence of ERα, whereas, in the absence of ERα, it can partially replace ERα. In conclusion, our in vivo data indicate that an important physiological role of ERβ is to modulate ERα-mediated gene transcription supporting a “Ying Yang” relationship between ERα and ERβ in mice.

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The AF-1 Activation Function of Estrogen Receptor α Is Necessary and Sufficient for Uterine Epithelial Cell Proliferation In Vivo

Endocrinology ◽

10.1210/en.2012-2059 ◽

2013 ◽

Vol 154 (6) ◽

pp. 2222-2233 ◽

Cited By ~ 42

Author(s):

Anne Abot ◽

Coralie Fontaine ◽

Isabelle Raymond-Letron ◽

Gilles Flouriot ◽

Marine Adlanmerini ◽

...

Keyword(s):

Cell Proliferation ◽

Estrogen Receptor ◽

Epithelial Cell ◽

Gene Transcription ◽

Crucial Role ◽

Estrogen Receptor Α ◽

Epithelial Cell Proliferation ◽

Wild Type

Abstract Estrogen receptor-α (ERα) regulates gene transcription through the 2 activation functions (AFs) AF-1 and AF-2. The crucial role of ERαAF-2 was previously demonstrated for endometrial proliferative action of 17β-estradiol (E2). Here, we investigated the role of ERαAF-1 in the regulation of gene transcription and cell proliferation in the uterus. We show that acute treatment with E2 or tamoxifen, which selectively activates ERαAF-1, similarly regulate the expression of a uterine set of estrogen-dependent genes as well as epithelial cell proliferation in the uterus of wild-type mice. These effects were abrogated in mice lacking ERαAF-1 (ERαAF-10). Four weeks of E2 treatment led to uterine hypertrophy and sustained luminal epithelial and stromal cell proliferation in wild-type mice, but not in ERαAF-10 mice. However, ERαAF-10 mice still presented a moderate uterine hypertrophy essentially due to a stromal edema, potentially due to the persistence of Vegf-a induction. Epithelial apoptosis is largely decreased in these ERαAF-10 uteri, and response to progesterone is also altered. Finally, E2-induced proliferation of an ERα-positive epithelial cancer cell line was also inhibited by overexpression of an inducible ERα isoform lacking AF-1. Altogether, these data highlight the crucial role of ERαAF-1 in the E2-induced proliferative response in vitro and in vivo. Because ERαAF-1 was previously reported to be dispensable for several E2 extrareproductive protective effects, an optimal ERα modulation could be obtained using molecules activating ERα with a minimal ERαAF-1 action.

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Both Estrogen Receptor α and β Stimulate Pituitary GH Gene Expression

Molecular Endocrinology ◽

10.1210/me.2013-1245 ◽

2014 ◽

Vol 28 (1) ◽

pp. 40-52 ◽

Cited By ~ 38

Author(s):

Dimiter Avtanski ◽

Horacio J. Novaira ◽

Sheng Wu ◽

Christopher J. Romero ◽

Rhonda Kineman ◽

...

Keyword(s):

Estrogen Receptor ◽

Mrna Expression ◽

Transcriptional Control ◽

Estrogen Receptor Α ◽

In Vitro Models ◽

Mrna Levels ◽

Ovariectomized Mice ◽

Serum Gh

Abstract Although sex steroids have been implicated in the control of mammalian growth, their direct effect on GH synthesis is less clear. The aim of this study was to establish whether estradiol (E2) directly affects GH synthesis in somatotrophs. Somatotroph GH3 and MtT/S cells were used as in vitro models. At physiological doses of E2 stimulation, GH mRNA levels were increased and the ER antagonist ICI 182,780 completely abolished this effect. Estrogen receptor (ER) α– and ERβ-selective agonists, propylpyrazole triol (PPT), and 2,3-bis(4-hydroxyphenyl) propionitrile (DPN), respectively, augmented GH mRNA expression and secretion, whereas E2 and PPT, but not DPN increased prolactin (PRL) mRNA levels. E2, PPT, and DPN stimulated expression of the pituitary transcription factor Pou1f1 and increased its binding to the GH promoter. In vivo evidence of E2 effects on GH synthesis was obtained from the generation of the somatotroph-specific ERα knockout (sERα-KO) mouse model. Basal pituitary GH, PRL, POU1F1, and ERα mRNA expression levels were lower in sERα-KO mice compared with those in controls; whereas ERβ mRNA levels remained unchanged. E2 and DPN stimulated pituitary GH mRNA expression and serum GH levels in control and sERα-KO ovariectomized mice; however, serum GH levels were unchanged in PPT-treated ovariectomized sERα-KO mice. In these animal models, PRL mRNA levels increased after either E2 or PPT, but an increase was not seen after DPN treatment. Thus, we propose a mechanism by which estrogen directly regulates somatotroph GH synthesis at a pretranslational level. In contrast to the predominant effect of ERα in the lactotroph, these results support a role for both ERα and ERβ in the transcriptional control of Gh in the somatotroph and illustrate important differences in ER isoform specificity in the anterior pituitary gland.

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Postlesion Estradiol Treatment Increases Cortical Cholinergic Innervations via Estrogen Receptor-α Dependent Nonclassical Estrogen Signaling in Vivo

Endocrinology ◽

10.1210/en.2011-1017 ◽

2011 ◽

Vol 152 (9) ◽

pp. 3471-3482 ◽

Cited By ~ 10

Author(s):

Zsombor Kőszegi ◽

Éva M. Szegő ◽

Rachel Y. Cheong ◽

Emeline Tolod-Kemp ◽

István M. Ábrahám

Keyword(s):

Estrogen Receptor ◽

Protein Kinase ◽

Cell Loss ◽

Estrogen Receptor Α ◽

Estrogen Signaling ◽

Ovariectomized Mice ◽

Cholinergic Cell ◽

Kinase A

17β-Estradiol (E2) treatment exerts rapid, nonclassical actions via intracellular signal transduction system in basal forebrain cholinergic (BFC) neurons in vivo. Here we examined the effect of E2 treatment on lesioned BFC neurons in ovariectomized mice and the role of E2-induced nonclassical action in this treatment. Mice given an N-methyl-d-aspartic acid (NMDA) injection into the substantia innominata-nucleus basalis magnocellularis complex (SI-NBM) exhibited cholinergic cell loss in the SI-NBM and ipsilateral cholinergic fiber loss in the cortex. A single injection of E2 after NMDA lesion did not have an effect on cholinergic cell loss in the SI-NBM, but it restored the ipsilateral cholinergic fiber density in the cortex in a time- and dose-dependent manner. The most effective cholinergic fiber restoration was observed with 33 ng/g E2 treatment at 1 h after NMDA lesion. The E2-induced cholinergic fiber restoration was absent in neuron-specific estrogen receptor-α knockout mice in vivo. Selective activation of nonclassical estrogen signaling in vivo by estren induced E2-like restorative actions. Selective blockade of the MAPK or protein kinase A pathway in vivo prevented E2's ability to restore cholinergic fiber loss. Finally, studies in intact female mice revealed an E2-induced restorative effect that was similar to that of E2-treated ovariectomized mice. These observations demonstrate that a single E2 treatment restores the BFC fiber loss in the cortex, regardless of endogenous E2 levels. They also reveal the critical role of nonclassical estrogen signaling via estrogen receptor-α and protein kinase A-MAPK pathways in E2-induced restorative action in the cholinergic system in vivo.

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Immunomodulation by 17β-estradiol in bivalve hemocytes

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00139.2006 ◽

2006 ◽

Vol 291 (3) ◽

pp. R664-R673 ◽

Cited By ~ 38

Author(s):

Laura Canesi ◽

Caterina Ciacci ◽

Lucia Cecilia Lorusso ◽

Michele Betti ◽

Tiziana Guarnieri ◽

...

Keyword(s):

Kinase Inhibitors ◽

Hydrolytic Enzymes ◽

Physiological Role ◽

Estrogen Receptor Α ◽

Nitrite Accumulation ◽

No Production ◽

17Β Estradiol

In mammals, estrogens have dose- and cell-type-specific effects on immune cells and may act as pro- and anti-inflammatory stimuli, depending on the setting. In the bivalve mollusc Mytilus, the natural estrogen 17β-estradiol (E2) has been shown to affect neuroimmune functions. We have investigated the immunomodulatory role of E2 in Mytilus hemocytes, the cells responsible for the innate immune response. E2 at 5–25 nM rapidly stimulated phagocytosis and oxyradical production in vitro; higher concentrations of E2 inhibited phagocytosis. E2-induced oxidative burst was prevented by the nitric oxide (NO) synthase inhibitor NG-monomethyl-l-arginine and superoxide dismutase, indicating involvement of NO and O2−; NO production was confirmed by nitrite accumulation. The effects of E2 were prevented by the antiestrogen tamoxifen and by specific kinase inhibitors, indicating a receptor-mediated mechanism and involvement of p38 MAPK and PKC. E2 induced rapid and transient increases in the phosphorylation state of PKC, as well as of a aCREB-like (cAMP responsive element binding protein) transcription factor, as indicated by Western blot analysis with specific anti-phospho-antibodies. Localization of estrogen receptor-α- and -β-like proteins in hemocytes was investigated by immunofluorescence confocal microscopy. The effects of E2 on immune function were also investigated in vivo at 6 and 24 h in hemocytes of E2-injected mussels. E2 significantly affected hemocyte lysosomal membrane stability, phagocytosis, and extracellular release of hydrolytic enzymes: lower concentrations of E2 resulted in immunostimulation, and higher concentrations were inhibitory. Our data indicate that the physiological role of E2 in immunomodulation is conserved from invertebrates to mammals.

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The Centrosomal, Putative Tumor Suppressor Protein TACC2 Is Dispensable for Normal Development, and Deficiency Does Not Lead to Cancer

Molecular and Cellular Biology ◽

10.1128/mcb.24.14.6403-6409.2004 ◽

2004 ◽

Vol 24 (14) ◽

pp. 6403-6409 ◽

Cited By ~ 28

Author(s):

Michael M. Schuendeln ◽

Roland P. Piekorz ◽

Christian Wichmann ◽

Youngsoo Lee ◽

Peter J. McKinnon ◽

...

Keyword(s):

Tumor Suppressor ◽

Cell Cycle Progression ◽

Tumor Development ◽

Coiled Coil ◽

Physiological Role ◽

Tumor Suppressor Protein ◽

Wild Type ◽

Mouse Development

ABSTRACT TACC2 is a member of the transforming acidic coiled-coil-containing protein family and is associated with the centrosome-spindle apparatus during cell cycling. In vivo, the TACC2 gene is expressed in various splice forms predominantly in postmitotic tissues, including heart, muscle, kidney, and brain. Studies of human breast cancer samples and cell lines suggest a putative role of TACC2 as a tumor suppressor protein. To analyze the physiological role of TACC2, we generated mice lacking TACC2. TACC2-deficient mice are viable, develop normally, are fertile, and lack phenotypic changes compared to wild-type mice. Furthermore, TACC2 deficiency does not lead to an increased incidence of tumor development. Finally, in TACC2-deficient embryonic fibroblasts, proliferation and cell cycle progression as well as centrosome numbers are comparable to those in wild-type cells. Therefore, TACC2 is not required, nonredundantly, for mouse development and normal cell proliferation and is not a tumor suppressor protein.

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Role of α2-macroglobulin in fever and cytokine responses induced by lipopolysaccharide in mice

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00746.2001 ◽

2002 ◽

Vol 283 (1) ◽

pp. R218-R226 ◽

Cited By ~ 24

Author(s):

Alexander V. Gourine ◽

Valery N. Gourine ◽

Yohannes Tesfaigzi ◽

Nathalie Caluwaerts ◽

Fred Van Leuven ◽

...

Keyword(s):

Mrna Level ◽

Physiological Role ◽

Mrna Levels ◽

Wild Type ◽

Cytokine Responses ◽

Α2 Macroglobulin ◽

Tnf Α ◽

Factor Α ◽

Necrosis Factor

α2-Macroglobulin (α2M) is not only a proteinase inhibitor in mammals, but it is also a specific cytokine carrier that binds pro- and anti-inflammatory cytokines implicated in fever, including interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α). To define the role of α2M in regulation of febrile and cytokine responses, wild-type mice and mice deficient in α2M (α2M −/−) were injected with lipopolysaccharide (LPS). Changes in body temperature as well as plasma levels of IL-1β, IL-6, and TNF-α and hepatic TNF-α mRNA level during fever in α2M −/− mice were compared with those in wild-type control mice. The α2M −/− mice developed a short-term markedly attenuated (ANOVA, P < 0.05) fever in response to LPS (2.5 mg/kg ip) compared with the wild-type mice. At 1.5 h after injection of LPS, the plasma concentration of TNF-α, but not IL-1β or IL-6, was significantly lower (by 58%) in the α2M −/− mice compared with their wild-type controls (ANOVA, P < 0.05). There was no difference in hepatic TNF-α mRNA levels between α2M −/− and wild-type mice 1.5 h after injection of LPS. These data support the hypotheses that 1) α2M is important for the normal development of LPS-induced fever and 2) a putative mechanism of α2M involvement in fever is through the inhibition of TNF-α clearance. These findings indicate a novel physiological role for α2M.

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Role of aspartate 351 in transactivation and active conformation of estrogen receptor α

Journal of Molecular Endocrinology ◽

10.1677/jme.1.01846 ◽

2005 ◽

Vol 35 (3) ◽

pp. 449-464 ◽

Cited By ~ 6

Author(s):

Jeong Hoon Kim ◽

Mee Hyun Lee ◽

Byoung Jin Kim ◽

Jun Hyun Kim ◽

Seong Jun Han ◽

...

Keyword(s):

Estrogen Receptor ◽

Dna Binding ◽

Transcriptional Activation ◽

Transcriptional Activity ◽

Steroid Receptors ◽

Estrogen Receptor Α ◽

Orphan Nuclear Receptor ◽

Active Conformation ◽

Wild Type

Estrogen-dependent transcriptional activation by estrogen receptor α (ERα) depends on the conformation of helices 3 and 12 in the ligand-binding domain. To better understand the function of helix 3 in ERα, we examined the role of charged residues, which are conserved in most steroid receptors in helix 3, in estrogen-dependent transactivation. The replacement of Asp-351 with lysine (D351K) or leucine (D351 L) completely abolished estrogen-dependent transactivation without affecting estrogen-binding, DNA-binding and homodimerization activities in ERα. The mutations dramatically reduced the ligand-dependent activation function 2 activity and impaired the ability of ERα to bind p160 coactivators. In addition, the D351K mutant effectively inhibited the transcriptional activation activity of wild-type ERα. Furthermore Asp-351 was required not only for the estrogen-dependent conformational change of wild-type ERα but also for the constitutive transcriptional activity and ligand-independent active conformation of ERα mutant Y537N. Similarly, in the orphan nuclear receptor called estrogen-related receptor 3 (ERR3), the replacement of Asp-273 (the corresponding amino acid to Asp-351 in ERα) with lysine abolished constitutive transcriptional activity of ERR3 without affecting DNA-binding activity and impaired the ability of the receptor to interact with p160 coactivators. These data suggest a role of Asp-351 in inducing and stabilizing the active conformation of ERα, and our results experimentally confirm the concept that Asp-351 in helix 3 interacts with the amide hydrogen of L540 in helix 12 to form a transcriptionally competent surface for binding p160 coactivators.

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C-Cbl Regulates c-Mpl Receptor Trafficking and Its Internalization

Blood ◽

10.1182/blood.v126.23.2388.2388 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2388-2388

Author(s):

Sebastian Jonas Saur ◽

Melanie Märklin ◽

Manuela Ganser ◽

Kyle Hoehn ◽

James E David ◽

...

Keyword(s):

Bone Marrow ◽

Plasma Levels ◽

Thrombus Formation ◽

Surface Expression ◽

Mrna Levels ◽

Wild Type ◽

Primary Mouse

Abstract Megakaryopoiesis is controlled by a variety of hematopoietic growth factors and cytokines in order to maintain physiological levels of circulating platelets. Thrombopoietin (TPO) signalling via its receptor c-Mpl is a key regulator of megakaryopoiesis driving megakaryocyte differentiation, promoting endomitosis and proplatelet formation. Therefore TPO/c-Mpl signalling needs to be tightly regulated to maintain physiological megakaryopoiesis. One of the most effective mechanisms to permanently disable activated signalling proteins is by targeted degradation via lysosomes or proteasomes. Previous studies have identified c-Cbl as an E3 ligase responsible for the ubiquitination of c-Mpl in cell lines. In this study, we investigated the mechanisms of TPO-mediated c-Mpl degradation in primary mouse cells. In order to determine the potential role of c-Cbl in murine megakaryopoiesis we used a conditional PF4-Cre c-Cbl knockout (ko) mouse model to specifically delete c-Cbl in the megakaryocytic lineage. Megakaryocytes were generated in vitro by culturing bone marrow from WT and PF4-Cre/c-Cbl-floxed (c-Cbl ko) lines for 72 hrs in the presence of rmTPO. C-Cbl ko mice showed significant bone marrow megakaryocyte hyperplasia, however megakaryocyte numbers in the spleen remained unchanged. Platelets counts were significantly elevated as compared to control mice (1.2 x106 vs. 1.7x106 p=0.0001) and in addition, the platelets from the c-Cbl ko mouse strain were of significantly smaller size (43 vs. 38 fL, p=0.0022). Using a method of in vivo double labelling of platelets, we were able to simultaneously follow the survival of both the entire population of platelets and new platelets which were generated during the last 24 hours. There were more new platelets produced within a 24 h period in the c-Cbl ko mice although the half-life of platelets was similar in the both cohorts. Although c-Cbl ko mice exhibited thrombocytosis, they showed a severe defect in thrombus formation using an in vivo thrombus formation model with Fe3Cl. TPO plasma levels, known to be inversely regulated by circulating platelet numbers, were surprisingly increased (250 vs. 420 pg/ml, p=0.005) in the c-Cbl ko mice. There was no difference in liver mRNA levels in the two cohorts. We therefore looked at c-Mpl protein and mRNA expression in megakaryocytes and found c-Cbl ko mice to express more c-Mpl compared with wild type controls. Surprisingly, we found c-Mpl surface expression to be reduced and internalization of the receptor significantly impaired following TPO stimulation in c-Cbl ko mice. Incubating platelets in vitro with TPO for 2 hours to evaluate the TPO uptake capacity of platelets, we found c-Cbl ko platelets to show a severe uptake defect compared with wild type control platelets. Taken together, we have successfully ablated c-Cbl specifically from the megakaryocyte lineage and demonstrated that this has profound effects on platelet counts and size. In addition, we showed that c-Cbl ablation leads to reduced c-Mpl surface expression and impaired internalization, which culminates in increased TPO plasma levels causing increased megakaryopoiesis in the c-Cbl ko mice. In summary, our data enhance our understanding of the regulation of TPO signalling and the physiological role of c-Cbl in the megakaryocytic lineage. Disclosures No relevant conflicts of interest to declare.

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Effects of Castration and Chronic Steroid Treatments on Hypothalamic Gonadotropin-Releasing Hormone Content and Pituitary Gonadotropins in Male Wild-Type and Estrogen Receptor-α Knockout Mice

Endocrinology ◽

10.1210/endo.139.10.6253 ◽

1998 ◽

Vol 139 (10) ◽

pp. 4092-4101 ◽

Cited By ~ 77

Author(s):

Jonathan Lindzey ◽

William C. Wetsel ◽

John F. Couse ◽

Tammy Stoker ◽

Ralph Cooper ◽

...

Keyword(s):

Estrogen Receptor ◽

Replacement Therapy ◽

Signaling Pathways ◽

Messenger Rna ◽

Estrogen Receptor Α ◽

Mrna Levels ◽

Wild Type ◽

Feedback Effects ◽

Serum Lh ◽

Circulating Levels

Abstract Testicular androgens are integral components of the hormonal feedback loops that regulate circulating levels of LH and FSH. The sites of feedback include hypothalamic areas regulating GnRH neurons and pituitary gonadotropes. To better define the roles of androgen receptor (AR), estrogen receptor-α (ERα), and estrogen receptor-β (ERβ) in mediating feedback effects of sex steroids on reproductive neuroendocrine function, we have determined the effects of castration and steroid replacement therapy on hypothalamic GnRH content, pituitary LHβ and FSHβ messenger RNA (mRNA) levels, and serum gonadotropins in male wild-type (WT) and estrogen receptor-α knockout (ERKO) mice. Hypothalami from intact WT and ERKO males contained similar amounts of GnRH, whereas castration significantly reduced GnRH contents in both genotypes. Replacement therapy with estradiol (E2), testosterone (T), or dihydrotestosterone (DHT) restored hypothalamic GnRH content in castrated (CAST) WT mice; only the androgens were effective in CAST ERKOs. Analyses of pituitary function revealed that LHβ mRNA and serum LH levels in intact ERKOs were 2-fold higher than those in intact WT males. Castration increased levels of LHβ mRNA (1.5- to 2-fold) and serum LH (4- to 5-fold) in both genotypes. Both E2 and T treatments significantly suppressed LHβ mRNA and serum LH levels in CAST WT males. However, E2 was completely ineffective, and T was only partially effective in suppressing these two indexes in the CAST ERKO males. DHT treatments stimulated a 50% increase in LHβ mRNA and serum LH levels in WT males, whereas serum LH was significantly suppressed in DHT-treated ERKO males. Although the pituitaries from intact ERKO males contained similar amounts of FSHβ mRNA, serum FSH levels were 20% higher than those in the intact WT males. Castration increased FSHβ mRNA levels only in WT males, but significantly increased serum FSH levels in both genotypes. Both E2 and T treatments significantly suppressed serum FSH in CAST WT males, whereas only E2 suppressed FSHβ mRNA. DHT treatments of CAST WT mice stimulated a small increase in serum FSH, but failed to alter FSHβ mRNA levels. None of the steroid treatments exerted any significant effect on FSHβ mRNA or serum FSH levels in CAST ERKOs. These data suggest that hypothalamic GnRH contents can be maintained solely through AR signaling pathways. However, normal regulation of gonadotrope function requires aromatization of T and activation of ERα signaling pathways in the gonadotrope. In addition, serum FSH levels in male ERKOs appear to be regulated largely by nonsteroidal testicular factors such as inhibin. Finally, these data suggest that hypothalamic ERβ may not be involved in mediating the negative feedback effects of T on serum LH and FSH in male mice.

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