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  • 1
    Keywords: RECEPTOR ; CANCER ; CANCER CELLS ; CELLS ; EXPRESSION ; IN-VITRO ; INHIBITOR ; CELL ; COMBINATION ; Germany ; INHIBITION ; KINASE ; VITRO ; GENE ; GENE-EXPRESSION ; GENES ; PROTEIN ; transcription ; METABOLISM ; ACCUMULATION ; ACTIVATION ; LIGAND ; TRANSCRIPTION FACTOR ; IMPACT ; REDUCTION ; DOWN-REGULATION ; PHOSPHORYLATION ; SIGNAL ; ALPHA ; BREAST ; breast cancer ; BREAST-CANCER ; ELEMENT ; RESPONSE ELEMENT ; TRANSCRIPTION FACTORS ; hormone ; gene expression ; ASSAY ; DECREASE ; CANCER-CELLS ; BETA ; transactivation ; DEGRADATION ; RECEPTORS ; PROTEASOME ; ESTRADIOL ; ESTROGEN-RECEPTOR ; CANCER CELL-LINES ; ENDOCRINE ; RE ; SYNTHASE ; PROTEIN-SYNTHESIS ; regulation ; REPORTER GENE ; SUBSTRATE ; ESTROGEN ; LEVEL ; analysis ; ASSAYS ; NUCLEAR ; estrogen receptor ; MCF-7 CELLS ; USA ; function ; HORMONES ; ANTAGONISTS ; PROTECTS ; progesterone receptor ; in combination ; E2 ; TURNOVER ; MESSENGER-RIBONUCLEIC-ACID
    Abstract: Glycogen synthase kinase- 3 ( GSK- 3) plays a key role in the regulation of transcription factors including steroid receptors. Having identified estrogen receptor-alpha ( ER alpha) as substrate for GSK- 3, the impact of GSK- 3 on ER alpha function and activity upon 17 beta- estradiol ( E2)- dependent activation remains to be clarified. Here we show by using small interfering technology in combination with immunoblot, gene expression analysis, and luciferase reporter assays that silencing of GSK- 3 alpha or GSK- 3 beta results in the reduction of ER alpha levels and transcriptional activity in ER alpha- positive breast cancer cells. Using MCF- 7 cells we demonstrate that reduction of ER alpha levels upon GSK- 3 silencing was due to increased proteasomal degradation of ER alpha rather than inhibition of ER alpha protein synthesis. Indeed, under this condition, ER alpha protein was rescued using the proteasome inhibitor MG132 in presence of the protein synthesis inhibitor cycloheximide. In addition, strong accumulation of ubiquitinated ER alpha was obtained after GSK- 3 silencing in the presence of MG132. We conclude that GSK- 3 protects ER alpha from proteasomal degradation and plays a crucial role in ER alpha protein stabilization and turnover. Furthermore, in vitro kinase assay depicted that GSK- 3 beta phosphorylates ER alpha at Ser- 118. GSK- 3 silencing resulted in decrease of E2- induced nuclear ER alpha phosphorylation at Ser- 118 and E2- induced estrogen response element- dependent luciferase reporter gene expression. Neither Ser- 118 phosphorylation nor luciferase activity was restored by use of MG132. Moreover, the expression of estrogenresponsive genes ( pS2 and progesterone receptor) was decreased upon GSK- 3 silencing. These findings demonstrated that GSK- 3 is required for E2- induced ER alpha phosphorylation at Ser118 and full transcriptional activity of the receptor upon E2 stimulation
    Type of Publication: Journal article published
    PubMed ID: 17609434
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  • 2
    Keywords: RECEPTOR ; EXPRESSION ; proliferation ; Germany ; IN-VIVO ; MODEL ; VIVO ; SUPPORT ; liver ; GENE ; GENE-EXPRESSION ; microarray ; transcription ; DRUG ; METABOLISM ; MICE ; ACTIVATION ; LIGAND ; DNA ; MECHANISM ; DOMAIN ; mechanisms ; BINDING ; DISCOVERY ; RECOGNITION ; ACID ; TARGET ; MOUSE ; gene expression ; MICROARRAY DATA ; DISRUPTION ; WOMEN ; DNA-BINDING ; EXCHANGE ; LIGANDS ; PHENOTYPE ; MOUSE MODEL ; GLUCOCORTICOID-RECEPTOR ; REPRESSION ; expression profiling ; ER ; ESTROGEN-RECEPTOR ; TRANSCRIPTIONAL REGULATION ; MAMMARY-GLAND ; regulation ; AMINO-ACID ; DRUG DISCOVERY ; MOUSE-LIVER ; development ; ESTROGEN ; LOCUS ; estrogen receptor ; USA ; DNA binding ; AGREEMENT ; BREAST-CANCER CELLS ; ESTROGENS ; POSITION ; RECEPTOR-ALPHA ; Genetic ; ALPHA GENE ; ER-ALPHA ; HORMONE-THERAPY ; MUTATION PROVIDES EVIDENCE
    Abstract: The majority of the biological effects of estrogens in the reproductive tract are mediated by estrogen receptor (ER)alpha, which regulates transcription by several mechanisms. Because the tissue-specific effects of some ER alpha ligands may be caused by tissue-specific transcriptional mechanisms of ER alpha, we aimed to identify the contribution of DNA recognition to these mechanisms in two clinically important target organs, namely uterus and liver. We used a genetic mouse model that dissects DNA binding-dependent vs. independent transcriptional regulation elicited by ER alpha. The EAAE mutant harbors amino acid exchanges at four positions of the DNA-binding domain (DBD) of ER alpha. This construct was knocked in the ER alpha gene locus to produce ER alpha((EAAE/ EAAE)) mice devoid of a functional ER alpha DBD. The phenotype of the ER alpha((EAAE/EAAE)) mice resembles the general loss-of-function phenotype of alpha ER knockout mutant mice with hypoplastic uteri, hemorrhagic ovaries, and impaired mammary gland development. In agreement with this phenotype, the expression pattern of the ER alpha((EAAE/EAAE)) mutant mice in liver obtained by genome-wide gene expression profiling supports the observation of a near-complete loss of estrogen-dependent gene regulation in comparison with the wild type. Further gene expression analyses to validate the results of the microarray data were performed by quantitative RT-PCR. The analyses indicate that both gene activation and repression by estrogen-bound ER alpha rely on an intact DBD in vivo. (Molecular Endocrinology 23: 1544-1555, 2009)
    Type of Publication: Journal article published
    PubMed ID: 19574448
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  • 3
    Keywords: PATHWAYS ; GENE-EXPRESSION ; ACTIVATION ; AMINO-ACIDS ; ER-ALPHA ; EPITHELIAL-CELL PROLIFERATION ; negative feedback ; RESPONSIVE ELEMENTS ; REPRODUCTIVE AXIS ; FEMALE MOUSE
    Abstract: Estrogen receptor alpha (ERalpha) interacts with DNA directly or indirectly via other transcription factors, referred to as "tethering." Evidence for tethering is based on in vitro studies and a widely used "KIKO" mouse model containing mutations that prevent direct estrogen response element DNA- binding. KIKO mice are infertile, due in part to the inability of estradiol (E2) to induce uterine epithelial proliferation. To elucidate the molecular events that prevent KIKO uterine growth, regulation of the pro-proliferative E2 target gene Klf4 and of Klf15, a progesterone (P4) target gene that opposes the pro-proliferative activity of KLF4, was evaluated. Klf4 induction was impaired in KIKO uteri; however, Klf15 was induced by E2 rather than by P4. Whole uterine chromatin immunoprecipitation-sequencing revealed enrichment of KIKO ERalpha binding to hormone response elements (HREs) motifs. KIKO binding to HRE motifs was verified using reporter gene and DNA-binding assays. Because the KIKO ERalpha has HRE DNA-binding activity, we evaluated the "EAAE" ERalpha, which has more severe DNA-binding domain mutations, and demonstrated a lack of estrogen response element or HRE reporter gene induction or DNA-binding. The EAAE mouse has an ERalpha null-like phenotype, with impaired uterine growth and transcriptional activity. Our findings demonstrate that the KIKO mouse model, which has been used by numerous investigators, cannot be used to establish biological functions for ERalpha tethering, because KIKO ERalpha effectively stimulates transcription using HRE motifs. The EAAE-ERalpha DNA-binding domain mutant mouse demonstrates that ERalpha DNA-binding is crucial for biological and transcriptional processes in reproductive tissues and that ERalpha tethering may not contribute to estrogen responsiveness in vivo.
    Type of Publication: Journal article published
    PubMed ID: 24713037
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  • 4
    Keywords: RECEPTOR ; EXPRESSION ; BLOOD ; CELL ; Germany ; IN-VIVO ; liver ; ENZYMES ; GENE ; GENES ; transcription ; METABOLISM ; MICE ; ACTIVATION ; kidney ; TRANSCRIPTION FACTOR ; INDUCTION ; hepatocytes ; MUTANT ; hormone ; DISRUPTION ; TANDEM MASS-SPECTROMETRY ; inactivation ; Jun ; GLUCOSE ; glucocorticoid receptor ; GLUCOCORTICOID-RECEPTOR ; ANTAGONIST ; insulin ; ABSENCE ; ADULT ; ENDOCRINE ; LEADS ; development ; CARBOXYKINASE GTP GENE ; HEPATIC GLUCONEOGENESIS ; PHOSPHOENOLPYRUVATE CARBOXYKINASE ; TYROSINE AMINOTRANSFERASE GENE
    Abstract: Hepatic glucose production by gluconeogenesis is the main source of glucose during fasting and contributes significantly to hyperglycemia in diabetes mellitus. Accordingly, glucose metabolism is tightly controlled by a variety of hormones including insulin, epinephrine, glucagon, and glucocorticoids (GCs) acting on various cell types. GC effects are mediated by the GC receptor (GR), a ligand-dependent transcription factor, which in the liver and kidney controls gluconeogenesis by induction of gluconeogenic enzymes. To specifically study the contribution of GC on liver carbohydrate metabolism, we generated mice with an inactivation of the GR gene exclusively in hepatocytes using the Cre/loxP technology. Half of the mutant mice die within the first 2 d after birth most likely due to hypoglycemia. Adult mice have normal blood sugar under basal conditions but show hypoglycemia after prolonged starvation due to reduced expression of genes involved in gluconeogenesis. We further demonstrate that absence of GR in hepatocytes limits the development of hyperglycemia in streptozotocin-induced diabetes mellitus probably due to impaired induction of gluconeogenesis. These findings show the essential role of GR function in liver glucose metabolism during fasting and in diabetic mice and indicate that liver-specific GC antagonists could be beneficial in control of diabetic hyperglycemia
    Type of Publication: Journal article published
    PubMed ID: 15031319
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  • 5
    Keywords: RECEPTOR ; APOPTOSIS ; CELLS ; EXPRESSION ; IN-VITRO ; proliferation ; SURVIVAL ; CELL-PROLIFERATION ; Germany ; IN-VIVO ; MODEL ; PATHWAYS ; VITRO ; GENERATION ; SYSTEM ; GENE ; GENE-EXPRESSION ; PROTEIN ; PROTEINS ; DIFFERENTIATION ; EPITHELIA ; MICE ; MOUSE ; gene expression ; DISRUPTION ; MUTATION ; EPITHELIAL-CELLS ; glucocorticoid receptor ; GLUCOCORTICOID-RECEPTOR ; ABSENCE ; DEFICIENCY ; RE ; secretion ; GLAND ; cell proliferation ; ACIDIC PROTEIN ; mineralocorticoid receptor ; EPITHELIUM ; mammary gland ; LACTATION ; GLAND DEVELOPMENT ; STAT5
    Abstract: Glucocorticoids have been shown to influence mammary gland function in vivo and to stimulate milk protein gene expression in vitro. Here, we describe the generation and analysis of a mouse model to study glucocorticoid receptor (GR, NR3C1) function in mammary epithelial cells. Using the Cre-loxP system, mutant mice were obtained in which the GR gene is specifically deleted in epithelial cells during lobuloalveolar development, leading to a complete loss of epithelial GR at the onset of lactation. Mice harboring the mammary-epithelial-specific GR mutation are able to nurse their litters until weaning. During pregnancy, however, GR deficiency delays lobuloalveolar development, leading to an incomplete epithelial penetration of the mammary fat pad that persists throughout lactation. We identified a reduced cell proliferation during lobuloalveolar development as reason for this delay. This reduction is compensated for by increased epithelial proliferation after parturition in the mutant glands. During lactation, GR-deficient mammary epithelium is capable of milk production and secretion. The expression of two milk proteins, namely whey acidic protein and beta-casein, during lactation was not critically affected in the absence of GR. We conclude that GR function is not essential for alveolar differentiation and milk production, but influences cell proliferation during lobuloalveolar development
    Type of Publication: Journal article published
    PubMed ID: 15471946
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  • 6
    Abstract: To study the role of glucocorticoid receptor (GR) at different stages of mammary gland development, mammary anlage were rescued from GR-/- mice by transplantation into the cleared fat pad of wild-type mice. In virgin mice, GR-/- outgrowths displayed abnormal ductal morphogenesis characterized by distended lumena, multiple layers of luminal epithelial cells in some regions along the ducts, and increased periductal stroma. In contrast, the loss of GR did not result in overt phenotypic changes in mammary gland development during pregnancy, lactation, and involution. Surprisingly, despite the known synergism between glucocorticoids and prolactin in the regulation of milk protein gene expression, whey acidic protein and beta-casein mRNA levels were unaffected in GR-/- transplants as compared with wild-type transplants. That mineralocorticoid receptor (MR) might compensate for the loss of GR was suggested by the detection of MR in the mammary gland at d 1 of lactation. This hypothesis was tested using explant cultures derived from the GR-/- transplants in which the mineralocorticoid fludrocortisone was able to synergistically induce beta-casein gene expression in the presence of prolactin and insulin. These studies suggest that MR may compensate for the absence of GR at some, but not at all stages of mammary gland development.
    Type of Publication: Journal article published
    PubMed ID: 12198239
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  • 7
    Keywords: brain ; PEPTIDE ; RECEPTOR ; CELLS ; EXPRESSION ; GROWTH ; INHIBITOR ; BLOOD ; CELL ; GENE ; GENES ; PROTEIN ; transcription ; METABOLISM ; MICE ; RELEASE ; ACTIVATION ; TRANSCRIPTION FACTOR ; IMPACT ; hepatocytes ; BINDING ; PHOSPHORYLATION ; SIGNAL ; ACID ; CREB ; ELEMENT-BINDING PROTEIN ; TRANSGENIC MICE ; hormone ; DISRUPTION ; BODY ; MUTANT MICE ; HYPOPLASIA ; BINDING PROTEIN ; LOSSES ; SIGNALS ; EXPANSION ; CAMP RESPONSE ELEMENT ; CREB FUNCTION ; HORMONE-RELEASING-HORMONE ; NEURAL STEM
    Abstract: The principal regulation of body growth is via a cascade of hormone signals emanating from the hypothalamus, by release of GHRH, which then directs the somatotroph cells of the pituitary to release GH into the blood stream. This in turn leads to activation of signal transducer and activator of transcription 5-dependent expression of genes such as IGF-1 in hepatocytes, acid labile substance, and serine protease inhibitor 2.1, resulting in body growth. Here, using conditional cAMP response element binding protein ( CREB) mutant mice, we show that loss of the CREB transcription factor in the brain, but not the pituitary, results in reduced postnatal growth consistent with dwarfism caused by GH deficiency. We demonstrate that although there appears to be no significant impact upon the expression of GHRH mRNA in CREB mutant mice, the amount of GHRH peptide is reduced. These findings show that CREB is required for the efficient production of GHRH in hypothalamus, in addition to its previously reported role in pituitary GH production and somatotroph expansion
    Type of Publication: Journal article published
    PubMed ID: 16141355
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  • 8
    Keywords: EXPRESSION ; SYSTEM ; EXPOSURE ; GENE ; GENE-EXPRESSION ; MICE ; DISRUPTION ; inactivation ; FIBRONECTIN ; VASCULAR DEVELOPMENT ; RECEPTOR GENE ; BRANCHING MORPHOGENESIS ; ENAC
    Abstract: Corticosteroid treatment is an established therapy for preterm infants, and germline inactivation of the glucocorticoid receptor (GR) gene in the mouse leads to respiratory failure and postnatal lethality. Although glucocorticoids have been thought to critically act in epithelial cells inducing the functional maturation of the lung, inactivation of the GR gene exclusively in the epithelium of the developing murine lung did not impair survival. In contrast, mice lacking GR specifically in mesenchyme-derived cells displayed a phenotype strongly reminiscent of GR knockout animals and died immediately after birth. Detailed analysis of gene expression allows the conclusion that GR acts in cells of the fibroblast lineage controlling their proliferation rate and the composition of the extracellular matrix.
    Type of Publication: Journal article published
    PubMed ID: 21659474
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  • 9
    Keywords: EXPRESSION ; SYSTEM ; MICE ; ACTIVATION ; INSULIN-RESISTANCE ; depression ; ANXIETY ; TARGETED MUTATIONS ; GENETIC DISSECTION ; ANTIDEPRESSANTS
    Abstract: The glucocorticoid receptor (GR) regulates hypothalamic-pituitary-adrenal (HPA) axis activity during the stress response. The paraventricular nucleus (PVN) is a major site of negative feedback to coordinate the degree of the HPA axis activity with the magnitude of the exposed stressor. To define the function of endogenous PVN GR, we used Cre-loxP technology to disrupt different GR exons in Sim1-expressing neurons of the hypothalamus. GR exon 2-deleted mice (Sim1Cre-GRe2Delta) demonstrated 43% loss of PVN GR compared with an 87% GR loss in exon 3-deleted mice (Sim1Cre-GRe3Delta). Sim1Cre-GRe3Delta mice display stunted growth at birth but develop obesity in adulthood and display impaired stress-induced glucose release. We observed elevated basal and stress-induced corticosterone levels in Sim1Cre-GRe3Delta mice, compared with control and Sim1Cre-GRe2Delta mice, and impaired dexamethasone suppression, indicating an inability to negatively regulate corticosterone secretion. Sim1Cre-GRe3Delta mice also showed increased CRH mRNA in the PVN, increased basal plasma ACTH levels, and reduced locomotor behavior. We observed no differences in Sim1Cre-GRe2Delta mice compared with control mice in any measure. Our behavioral data suggest that GR deletion in Sim1-expressing neurons has no effect on anxiety or despair-like behavior under basal conditions. We conclude that loss of PVN GR results in severe HPA axis hyperactivity and Cushing's syndrome-like phenotype but does not affect anxiety and despair-like behaviors.
    Type of Publication: Journal article published
    PubMed ID: 23979842
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