Dexamethasone represses cAMP rapid upregulation of TRH gene transcription: identification of a composite glucocorticoid response element and a cAMP response element in TRH promoter

Hypothalamic proTRH mRNA levels are rapidly increased (at 1 h) in vivo by cold exposure or suckling, and in vitro by 8Br-cAMP or glucocorticoids. The aim of this work was to study whether these effects occurred at the transcriptional level. Hypothalamic cells transfected with rat TRH promoter (− 776/+85) linked to the luciferase reporter showed increased transcription by protein kinase (PK) A and PKC activators, or by dexamethasone (dex), but co-incubation with dex and 8Br-cAMP decreased their stimulatory effect (as observed for proTRH mRNA levels). These effects were also observed in NIH-3T3-transfected cells supporting a characteristic of TRH promoter and not of hypothalamic cells. Transcriptional regulation by 8Br-cAMP was mimicked by noradrenaline which increased proTRH mRNA levels, but not in the presence of dex. PKA inhibition by H89 avoided 8Br-cAMP or noradrenaline stimulation. TRH promoter sequences, cAMP response element (CRE)-like (− 101/− 94 and − 59/− 52) and glucocorticoid response element (GRE) half-site (− 210/− 205), were analyzed by electrophoretic mobility shift assays with nuclear extracts from hypothalamic or neuroblastoma cultures. PKA stimulation increased binding to CRE (− 101/− 94) but not to CRE (− 59/− 52); dex or 12-O-tetradecanoylphorbol-13-acetate (TPA) increased binding to GRE, a composite site flanked by a perfect and an imperfect activator protein (AP-1) site in the complementary strand. Interference was observed in the binding of CRE or GRE with nuclear extracts from cells co-incubated for 3 h with 8Br-cAMP and dex; from cells incubated for 1 h, only the binding to GRE showed interference. Rapid cross-talk of glucocorticoids with PKA signaling pathways regulating TRH transcription constitutes another example of neuroendocrine integration.

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Identification of a functional glucocorticoid response element in the promoter of the cyclin-dependent kinase inhibitor p57Kip2

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0300359 ◽

2003 ◽

Vol 30 (3) ◽

pp. 359-368 ◽

Cited By ~ 24

Author(s):

K Alheim ◽

J Corness ◽

MK Samuelsson ◽

LG Bladh ◽

T Murata ◽

...

Keyword(s):

Glucocorticoid Receptor ◽

Kinase Inhibitor ◽

De Novo ◽

Cyclin Dependent Kinase ◽

Mobility Shift ◽

Sequence Comparisons ◽

Glucocorticoid Treatment ◽

Glucocorticoid Response Element ◽

Response Element ◽

Glucocorticoid Response

Glucocorticoids are known regulators of the cell cycle, normally exerting an anti-proliferative effect. We have previously shown that glucocorticoids stimulate expression of p57(Kip2), a member of the Cip/Kip family of cyclin-dependent kinase inhibitors which, in some cell types, may account for the anti-proliferative responses seen after glucocorticoid treatment. The induction of p57(Kip2) involves primary transcriptional effects where no de novo protein synthesis is necessary, suggesting a direct interaction of the glucocorticoid receptor with the p57(Kip2) gene. In this study we have identified a functional glucocorticoid response element (GRE), located 5 kilo bases (kb) upstream of the transcription start site in the human p57(Kip2) promoter. This GRE was functional also when isolated, suggesting a direct transcriptional effect of the glucocorticoid receptor. Furthermore, mutation of this GRE abolished glucocorticoid induction of the reporter gene, whereas mutation of a nearby Sp1 site did not. Using electrophoretic mobility shift assays, we have shown that the -5 kb p57(Kip2) promoter GRE was able to compete with a well-known GRE for glucocorticoid receptor binding. Sequence comparisons with the mouse genome showed that this GRE is highly conserved, further strengthening the biological importance of this site. All these data emphasize the involvement of this GRE in the glucocorticoid-mediated induction of p57(Kip2) expression.

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Glucocorticoids Stimulate Hepatic and Renal Catecholamine Inactivation by Direct Rapid Induction of the Dopamine Sulfotransferase Sult1d1

Endocrinology ◽

10.1210/en.2009-0590 ◽

2010 ◽

Vol 151 (1) ◽

pp. 185-194 ◽

Cited By ~ 16

Author(s):

Stephen Wong ◽

Kheng Tan ◽

Kirstyn T. Carey ◽

Atsushi Fukushima ◽

Tony Tiganis ◽

...

Keyword(s):

Stress Response ◽

Target Genes ◽

Collecting Duct ◽

Detoxification Enzymes ◽

Mrna Levels ◽

Glucocorticoid Response Element ◽

Response Element ◽

Endogenous Dopamine ◽

Glucocorticoid Response ◽

Primary Mouse

Abstract During the stress response and metabolic fasting, glucocorticoids acting via the glucocorticoid receptor (GR) stimulate hepatic glucose production by activating specific gluconeogenic enzyme target genes. To characterize novel direct GR-regulated hepatic target genes under glucocorticoid control, we performed a whole genome gene expression microarray using dexamethasone-treated GR-null mice. Strongly induced previously characterized genes included phosphoenolpyruvate carboxykinase, serine dehydratase, tyrosine oxygenase, lipin 1, metallothionine, and cdkn1A. Novel induced genes included Ddit4, Fkbp5, Megf9, Sult1e1, and Sult1d1, and all were verified by real-time PCR. Sult1d1, a sulfotransferase, is a member of a large superfamily of detoxification enzymes and has an important role in the inactivation of endogenous dopamine-derived compounds, including the catecholamines. Treatment of primary mouse hepatocytes with dexamethasone for 6 h dramatically increased Sult1d1 mRNA levels, whereas cotreatment with RU-486, a GR antagonist, blocked induction by dexamethasone. Sult1d1 mRNA levels were also increased by dexamethasone in the kidney, a major site of Sult1d1 synthesis. Sult1d1 mRNA was localized by in situ hybridization to renal collecting ducts and was rapidly induced by glucocorticoids in renal inner medullary collecting duct (IMCD3) cells. Hepatic and renal Sult1d1 enzymatic activity was significantly induced in vivo in wild-type mice 6 h after dexamethasone treatment. Chromatin immunoprecipitation assay analysis upstream of the Sult1d1 gene promoter identified a glucocorticoid response element close to the neighboring glucocorticoid-responsive estrogen sulfotransferase Sult1e1 gene, indicating that both genes potentially share a common glucocorticoid response element. These results suggest that Sult1d1 in mice is directly induced by glucocorticoids and may attenuate elevated catecholamine activity during the stress response.

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Temporal Recruitment of Transcription Factors at the 3′,5′-Cyclic Adenosine 5′-Monophosphate-Response Element of the Human GnRH-II Promoter

Endocrinology ◽

10.1210/en.2008-0481 ◽

2008 ◽

Vol 149 (10) ◽

pp. 5162-5171 ◽

Cited By ~ 4

Author(s):

Song Ling Poon ◽

Beum-Soo An ◽

Wai-Kin So ◽

Geoffrey L. Hammond ◽

Peter C. K. Leung

Keyword(s):

Transcription Factors ◽

Chromatin Immunoprecipitation ◽

Binding Protein ◽

Camp Response Element Binding ◽

Mrna Levels ◽

Camp Response Element ◽

Response Element ◽

Creb Phosphorylation ◽

Nuclear Extracts ◽

Camp Stimulation

GnRH-II is a potent GnRH subtype involved in modulating OVCAR-3 cell proliferation and the invasive properties of JEG-3 cells, and an atypical cAMP-response element (CRE) in the human GnRH-II promoter influences its activation. We demonstrated that the GnRH-II promoter is activated by 8-bromoadenosine-cAMP in several cell lines including αT3, TE671, JEG-3, and OVCAR-3 cells and that cAMP enhances GnRH-II mRNA levels in JEG-3 and OVCAR-3 cells. Moreover, 8-bromoadenosine-cAMP increases cAMP response element-binding protein (CREB) phosphorylation in JEG-3 and OVCAR-3 cells and augments CBP and CCAAT/enhancer-binding protein (C/EBP)-β coimmunoprecipitation with phosphorylated CREB (p-CREB) in a temporally defined manner from nuclear extracts. When CREB, CBP, and C/EBPβ levels were knocked down by small interfering RNA, reductions in any of these transcription factors reduced cAMP-enhanced GnRH-II promoter activity and GnRH-II mRNA levels in JEG-3 and OVCAR-3 cells. Importantly, chromatin immunoprecipitation assay showed that p-CREB bound the CRE within the endogenous GnRH-II promoter within 1 h and that p-CREB association with C/EBPβ occurs within 2 h of cAMP stimulation, coincident with the first appearance of C/EBPβ at the CRE. By contrast, maximum interactions between p-CREB and CBP do not occur until at least 4 h after cAMP stimulation, and this is reflected in the progressive loading of CBP at the CRE at 2–4 h, as demonstrated by chromatin immunoprecipitation. Taken together, these data suggest that p-CREB, C/EBPβ, and CBP are recruited to the CRE of the GnRH-II promoter in a temporarily defined manner to enhance its transcription in JEG-3 and OVCAR-3 cells in response to cAMP.

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Function of a truncated glucocorticoid receptor form at a negative glucocorticoid response element in the proopiomelanocortin gene

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0260043 ◽

2001 ◽

Vol 26 (1) ◽

pp. 43-49 ◽

Cited By ~ 9

Author(s):

MK Turney ◽

WJ Kovacs

Keyword(s):

Gene Expression ◽

Glucocorticoid Receptor ◽

Reporter Gene ◽

Reporter Gene Expression ◽

Orphan Nuclear Receptor ◽

Mobility Shift ◽

Glucocorticoid Response Element ◽

Response Element ◽

Glucocorticoid Response ◽

Regulatory Effects

ACTH-producing tumors of nonpituitary origin characteristically exhibit insensitivity to the negative feedback effects of glucocorticoids. In the DMS-79 cell line derived from an ACTH-producing small cell lung cancer we have previously identified an aberrantly spliced glucocorticoid receptor (GRDelta) that lacks a ligand-binding domain. We examined the interactions of this truncated form of GR with the proximal human proopiomelanocortin (POMC) promoter. In electrophoretic mobility shift assays GRDelta bound to the negative glucocorticoid response element (nGRE) at position -78 to -50 in the human POMC promoter. Nur77, an orphan nuclear receptor that exerts positive regulatory effects on the POMC gene is also known to bind to this DNA element. The functional properties of GR and GRDelta binding to this DNA element were examined in transient transfection experiments in murine AtT-20 corticotroph tumor cells. Reporter gene expression under the control of proximal POMC promoter elements was stimulated by addition of forskolin to the culture medium or by transfection with expression constructs for human Nak1, the human homologue of Nur77. Treatment of transfected cells with dexamethasone resulted in suppression of forskolin- or Nak1-stimulated POMC-reporter gene expression in the presence of co-transfected GR but not with GRDelta. The experiments indicate that in the human POMC promoter GRDelta is capable of binding to the nGRE but cannot effect trans-repression of POMC-reporter gene expression.

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A Glucocorticoid Response Element Enhances Transcription of a Methionine tRNA Gene inCisandTrans*

Molecular Endocrinology ◽

10.1210/mend-4-8-1173 ◽

1990 ◽

Vol 4 (8) ◽

pp. 1173-1182 ◽

Cited By ~ 2

Author(s):

Hsiao-Lin Li ◽

Eric B. Kmiec

Keyword(s):

Trna Gene ◽

Glucocorticoid Response Element ◽

Response Element ◽

Glucocorticoid Response

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Identification of a Functional Glucocorticoid Response Element in theCYP3A1/IGC2Gene

DNA and Cell Biology ◽

10.1089/dna.1998.17.39 ◽

1998 ◽

Vol 17 (1) ◽

pp. 39-49 ◽

Cited By ~ 17

Author(s):

TERESA M. PEREIRA ◽

JAN CARLSTEDT-DUKE ◽

M. CELESTE LECHNER ◽

JAN-ÅKE GUSTAFSSON

Keyword(s):

Glucocorticoid Response Element ◽

Response Element ◽

Glucocorticoid Response

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Enhancement by lithium of cAMP-induced CRE/CREB-directed gene transcription conferred by TORC on the CREB basic leucine zipper domain

Biochemical Journal ◽

10.1042/bj20070796 ◽

2007 ◽

Vol 408 (1) ◽

pp. 69-77 ◽

Cited By ~ 24

Author(s):

Ulrike Böer ◽

Julia Eglins ◽

Doris Krause ◽

Susanne Schnell ◽

Christof Schöfl ◽

...

Keyword(s):

Dna Binding ◽

Gene Transcription ◽

Leucine Zipper ◽

Luciferase Reporter ◽

Lithium Salts ◽

Transactivation Domain ◽

Camp Response Element ◽

Basic Leucine Zipper ◽

Response Element ◽

Leucine Zipper Domain

The molecular mechanism of the action of lithium salts in the treatment of bipolar disorder is not well understood. As their therapeutic action requires chronic treatment, adaptive neuronal processes are suggested to be involved. The molecular basis of this are changes in gene expression regulated by transcription factors such as CREB (cAMP-response-element-binding protein). CREB contains a transactivation domain, in which Ser119 is phosphorylated upon activation, and a bZip (basic leucine zipper domain). The bZip is involved in CREB dimerization and DNA-binding, but also contributes to CREB transactivation by recruiting the coactivator TORC (transducer of regulated CREB). In the present study, the effect of lithium on CRE (cAMP response element)/CREB-directed gene transcription was investigated. Electrically excitable cells were transfected with CRE/CREB-driven luciferase reporter genes. LiCl (6 mM or higher) induced an up to 4.7-fold increase in 8-bromo-cAMP-stimulated CRE/CREB-directed transcription. This increase was not due to enhanced Ser119 phosphorylation or DNA-binding of CREB. Also, the known targets inositol monophosphatase and GSK3β (glycogen-synthase-kinase 3β) were not involved as specific GSK3β inhibitors and inositol replenishment did not mimic and abolish respectively the effect of lithium. However, lithium no longer enhanced CREB activity when the CREB-bZip was deleted or the TORC-binding site inside the CREB-bZip was specifically mutated (CREB-R300A). Otherwise, TORC overexpression conferred lithium responsiveness on CREB-bZip or the CRE-containing truncated rat somatostatin promoter. This indicates that lithium enhances cAMP-induced CRE/CREB-directed transcription, conferred by TORC on the CREB-bZip. We thus support the hypothesis that lithium salts modulate CRE/CREB-dependent gene transcription and suggest the CREB coactivator TORC as a new molecular target of lithium.

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