scholarly journals The methylated-DNA binding protein MBD2 enhances NGFI-A (egr-1)-mediated transcriptional activation of the glucocorticoid receptor

2014 ◽  
Vol 369 (1652) ◽  
pp. 20130513 ◽  
Author(s):  
Ian C. G. Weaver ◽  
Ian C. Hellstrom ◽  
Shelley E. Brown ◽  
Stephen D. Andrews ◽  
Sergiy Dymov ◽  
...  
Keyword(s):  
Glucocorticoid Receptor ◽  
Transcriptional Activation ◽  
Target Genes ◽  
Protein A ◽  
Site Directed Mutagenesis ◽  
Signalling Pathways ◽  
Hek 293 ◽  
293 Cells ◽  
Exon 1 ◽  
Inducible Protein

Variations in maternal care in the rat influence the epigenetic state and transcriptional activity of glucocorticoid receptor (GR) gene in the hippocampus. The mechanisms underlying this maternal effect remained to be defined, including the nature of the relevant maternally regulated intracellular signalling pathways. We show here that increased maternal licking/grooming (LG), which stably enhances hippocampal GR expression, paradoxically increases hippocampal expression of the methyl-CpG binding domain protein-2 (MBD2) and MBD2 binding to the exon 1 7 GR promoter. Knockdown experiments of MBD2 in hippocampal primary cell culture show that MBD2 is required for activation of exon 1 7 GR promoter. Ectopic co-expression of nerve growth factor-inducible protein A (NGFI-A) with MBD2 in HEK 293 cells with site-directed mutagenesis of the NGFI-A response element within the methylated exon 1 7 GR promoter supports the hypothesis that MBD2 collaborates with NGFI-A in binding and activation of this promoter. These data suggest a possible mechanism linking signalling pathways, which are activated by behavioural stimuli and activation of target genes.

scholarly journals Glucocorticoid Receptor Phosphorylation Differentially Affects Target Gene Expression

2008 ◽  
Vol 22 (8) ◽  
pp. 1754-1766 ◽  
Author(s):  
Weiwei Chen ◽  
Thoa Dang ◽  
Raymond D. Blind ◽  
Zhen Wang ◽  
Claudio N. Cavasotto ◽  
...  
Keyword(s):  
Glucocorticoid Receptor ◽  
Transcriptional Activation ◽  
Leucine Zipper ◽  
Target Genes ◽  
Divalent Cations ◽  
Phosphorylation Site ◽  
Transcriptional Response ◽  
Receptor Occupancy ◽  
Wild Type ◽  
Interacting Protein

Abstract The glucocorticoid receptor (GR) is phosphorylated at multiple sites within its N terminus (S203, S211, S226), yet the role of phosphorylation in receptor function is not understood. Using a range of agonists and GR phosphorylation site-specific antibodies, we demonstrated that GR transcriptional activation is greatest when the relative phosphorylation of S211 exceeds that of S226. Consistent with this finding, a replacement of S226 with an alanine enhances GR transcriptional response. Using a battery of compounds that perturb different signaling pathways, we found that BAPTA-AM, a chelator of intracellular divalent cations, and curcumin, a natural product with antiinflammatory properties, reduced hormone-dependent phosphorylation at S211. This change in GR phosphorylation was associated with its decreased nuclear retention and transcriptional activation. Molecular modeling suggests that GR S211 phosphorylation promotes a conformational change, which exposes a novel surface potentially facilitating cofactor interaction. Indeed, S211 phosphorylation enhances GR interaction with MED14 (vitamin D receptor interacting protein 150). Interestingly, in U2OS cells expressing a nonphosphorylated GR mutant S211A, the expression of IGF-binding protein 1 and interferon regulatory factor 8, both MED14-dependent GR target genes, was reduced relative to cells expressing wild-type receptor across a broad range of hormone concentrations. In contrast, the induction of glucocorticoid-induced leucine zipper, a MED14-independent GR target, was similar in S211A- and wild-type GR-expressing cells at high hormone levels, but was reduced in S211A cells at low hormone concentrations, suggesting a link between GR phosphorylation, MED14 involvement, and receptor occupancy. Phosphorylation also affected the magnitude of repression by GR in a gene-selective manner. Thus, GR phosphorylation at S211 and S226 determines GR transcriptional response by modifying cofactor interaction. Furthermore, the effect of GR S211 phosphorylation is gene specific and, in some cases, dependent upon the amount of activated receptor.

Splice variants of a ClC-2 chloride channel with differing functional characteristics

2000 ◽  
Vol 279 (4) ◽  
pp. C1198-C1210 ◽  
Author(s):  
L. Pablo Cid ◽  
María-Isabel Niemeyer ◽  
Alfredo Ramírez ◽  
Francisco V. Sepúlveda
Keyword(s):  
Splice Variants ◽  
Donor Site ◽  
Proximal Colon ◽  
Site Directed Mutagenesis ◽  
Intestinal Epithelial ◽  
Chloride Currents ◽  
Hek 293 ◽  
Internal Donor ◽  
293 Cells ◽  
Functional Consequences

We identified two ClC-2 clones in a guinea pig intestinal epithelial cDNA library, one of which carries a 30-bp deletion in the NH2 terminus. PCR using primers encompassing the deletion gave two products that furthermore were amplified with specific primers confirming their authenticity. The corresponding genomic DNA sequence gave a structure of three exons and two introns. An internal donor site occurring within one of the exons accounts for the deletion, consistent with alternative splicing. Expression of the variants gpClC-2 and gpClC-2Δ77–86 in HEK-293 cells generated inwardly rectifying chloride currents with similar activation characteristics. Deactivation, however, occurred with faster kinetics in gpClC-2Δ77–86. Site-directed mutagenesis suggests that a protein kinase C-mediated phosphorylation consensus site lost in gpClC-2Δ77–86 is not responsible for the observed change. The deletion-carrying variant is found in most tissues examined, and it appears more abundant in proximal colon, kidney, and testis. The presence of a splice variant of ClC-2 modified in its NH2-terminal domain could have functional consequences in tissues where their relative expression levels are different.

Analysis of MLL Amplification and 5q Deletions in AML/MDS Cell Lines Identifies Novel Targets.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1424-1424
Author(s):  
Bjoern Schneider ◽  
Stefan Nagel ◽  
Maren Kaufmann ◽  
Hilmar Quentmeier ◽  
Yoshinobu Matsuo ◽  
...  
Keyword(s):  
Cell Lines ◽  
Transcriptional Activation ◽  
Myeloid Leukemia ◽  
Target Genes ◽  
Histone Acetyltransferase ◽  
Cell Types ◽  
Signalling Pathways ◽  
Specific Cell ◽  
Genomic Amplification ◽  
The Common

Abstract Genomic amplifications of the 11q23 region occur in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) where MLL and a few neighboring genes, notably DDX6, are deemed salient targets. However, the extent to which amp(lified)-MLL and translocated MLL share effector targets remains to be established. Even less is known about the target(s) of deletions affecting the long arm of chromosome 5 (5q-) which reportedly partner amp-MLL. We analyzed three AML/MDS cell lines by cytogenetics (conventional and FISH) in parallel with real time q(uantitative)-PCR at both 11q23 and 5q2 to measure copy number and expression of salient target genes together with putative downstream targets. The cell lines comprised: MOLM-17 (transforming-MDS), SAML-2 (therapy-related AML), and UOC-M1 (AML-M1). All three cell lines exhibited approximately four-fold genomic amplification of 11q23 including MLL and DDX6, while the amplicon extended telomerically to include FLI1 (11q24) and HNT (11q25) in MOLM-17 and UOC-M1 only. Expression, quantified relative to AML/MDS cell lines without MLL rearrangement, revealed that of the genomically amplified genes only MLL was generally overexpressed, namely by 9.5x (MOLM-17), 5.1x (UOC-M1), and 4.6x (SAML-2). In addition to the highest MLL expression, in MOLM-17 FLI1 (3.8x) and DDX6 (2.8x) were significantly upregulated. Expression was also quantified among reputed MLL target genes, and showed that in the three cell lines MEIS1 was upregulated in MOLM-17 only (by 6x), and CDKN2C in all cell lines (by about 2x), while HOXA9 and CDKN1B showed near-normal levels of expression. All three cell lines carried 5q- with a common deleted region at 5q31 extending from 134.2–137.5 Mbp. Of a panel of genes recently identified as 5q- deletion targets (centromere-TIGA1, CAMLG, C5orf15, C5orf14, BRD8, HARS, KIAA0141, CSNK1A1, RBM22-telomere), only C5orf15 (function unknown) and BRD8 (a component of the nua4 histone acetyltransferase complex involved in transcriptional activation) were generally downregulated - to about 0.25x, and about 0.4x normalized expression levels, respectively. Both genes lie within the common deleted region. In summary, we have characterized amp-MLL and 5q- in MOLM-17, the first MDS cell line to be described with these rearrangements, together with two AML cell lines with similar cytogenetic profiles. Our data suggest that MLL is the only clear object of 11q23 amplification hitherto identified and CDKN2C its sole unequivocal target in AML/MDS cell lines. It is possible that MEIS1 is also targeted for activation in specific cell types or disease phases in MDS. These findings also highlight C5orf15 and/or BRD8 as possible leukemogenic accomplices targeted for downregulation in accompanying 5q-. These findings may point to differences in signalling pathways targeted by amp-MLL in AML and MDS.

The Impact of Glu102Lys on the Factor X Function in a Patient with a Doubly Homozygous Factor X Deficiency (Gla14Lys and Glu102Lys)

2000 ◽  
Vol 83 (02) ◽  
pp. 234-238 ◽  
Author(s):  
Eva Forberg ◽  
Iris Huhmann ◽  
Ester Jimenez-Boj ◽  
Herbert Watzke
Keyword(s):  
Point Mutations ◽  
Site Directed Mutagenesis ◽  
Normal System ◽  
Factor X ◽  
Wild Type ◽  
Hek 293 ◽  
293 Cells ◽  
Functional Defect ◽  
A Minor ◽  
The Impact

SummaryTwo homozygous point mutations were found in a patient with factor X (FX) deficiency; One results in substitution of Lys for Gla+14 and the second causes a Lys substitution for Glu102. The proposita has a severely reduced FX coagulant activity in the extrinsic (<1% of normal) and in the intrinsic (30% of normal) system of coagulation and after activation with Russel’s viper venom (18% of normal). The FX antigen is reduced in this patient to 20% of normal. The substitution of Lys for Glu102 in FX deficiency has been reported previously in a heterozygous state in conjunction with a Lys for Gla+14 substitution and with a Pro for Ser334 substitution. The contribution of the Lys for Glu102 substitution in the observed combined FX defect in these patients was unclear. The mutation causing the Glu102Lys substitution was introduced by site directed mutagenesis into a wild-type FX cDNA, and recombinant protein was expressed in HEK 293 cells. Compared to the wild-type FX cDNA, the mutant construct had a 67% activity upon activation in the extrinsic system, 93% activity upon activation in the intrinsic system and 72% after activation with RVV. The data presented show that the substitution of Lys for Glu102 results in a minor functional defect of the FX molecule.

Cloning of Trp1β isoform from rat brain: immunodetection and localization of the endogenous Trp1 protein

1999 ◽  
Vol 276 (4) ◽  
pp. C969-C979 ◽  
Author(s):  
Weiching Wang ◽  
Brian O’Connell ◽  
Raymond Dykeman ◽  
Takayuki Sakai ◽  
Christine Delporte ◽  
...  
Keyword(s):  
Amino Acids ◽  
Rat Brain ◽  
Protein A ◽  
Protein S ◽  
Polyclonal Antiserum ◽  
Cell Fractionation ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Mouse Tissues

The Trp gene product has been proposed as a candidate protein for the store-operated Ca2+channel, but the Trp protein(s) has not been identified in any nonexcitable cell. We report here the cloning of a rat brain Trp1β cDNA and detection and immunolocalization of the endogenous and expressed Trp1 protein. A 400-bp product, with >95% homology to mouse Trp1, was amplified from rat submandibular gland RNA. Rat-specific primers were used for cloning of a full-length rat brain Trp1β cDNA (rTrp1), encoding a protein of 759 amino acids. Northern blot analysis demonstrated the transcript in several rat and mouse tissues. The peptide (amino acids 523–536) was used to generate a polyclonal antiserum. The affinity-purified antibody 1) immunoprecipitated human Trp1 (hTrp1) from transfected HEK-293 cells, 2) reacted with a protein of ∼92 kDa, but not with hTrp3, in membranes of hTrp3-expressing HEK-293 cells, and 3) reacted with proteins of 92 and 56 kDa in human and rat brain membranes. Confocal microscopy and cell fractionation demonstrated that endogenous and expressed hTrp1 and expressed hTrp3 proteins were localized in the plasma membrane of HEK-293 cells, consistent with their proposed role in Ca2+ influx. The data demonstrate for the first time the presence of Trp1 protein in a nonexcitable cell.

Activation and stabilization of human tryptophan hydroxylase 2 by phosphorylation and 14-3-3 binding

2008 ◽  
Vol 410 (1) ◽  
pp. 195-204 ◽  
Author(s):  
Ingeborg Winge ◽  
Jeffrey A. Mckinney ◽  
Ming Ying ◽  
Clive S. D'Santos ◽  
Rune Kleppe ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Tryptophan Hydroxylase ◽  
Enzyme Stability ◽  
Enzyme Activation ◽  
Site Directed Mutagenesis ◽  
Peripheral Tissues ◽  
Hek 293 ◽  
Rate Limiting Step ◽  
293 Cells ◽  
Dependent Protein

TPH (tryptophan hydroxylase) catalyses the rate-limiting step in the synthesis of serotonin, and exists in two isoforms: TPH1, mainly found in peripheral tissues and the pineal body, and TPH2, a neuronal form. In the present study human TPH2 was expressed in Escherichia coli and in HEK (human embryonic kidney)-293 cells and phosphorylated using several different mammalian protein kinases. TPH2 was rapidly phosphorylated to a stoichiometry of 2 mol of phosphate/mol of subunit by PKA (protein kinase A), but only to a stoichiometry of 0.2 by Ca2+/calmodulin dependent protein kinase II. Both kinases phosphorylated Ser19, but PKA also phosphorylated Ser104, as determined by MS, phosphospecific antibodies and site-directed mutagenesis of several possible phosphorylation sites, i.e. Ser19, Ser99, Ser104 and Ser306. On average, purified TPH2 WT (wild-type) was activated by 30% after PKA phosphorylation and studies of the mutant enzymes showed that enzyme activation was mainly due to phosphorylation at Ser19. This site was phosphorylated to a stoichiometry of up to 50% in HEK-293 cells expressing TPH2, and the enzyme activity and phosphorylation stoichiometry was further increased upon treatment with forskolin. Purified PKA-phosphorylated TPH2 bound to the 14-3-3 proteins γ, ϵ and BMH1 with high affinity, causing a further increase in enzyme stability and activity. This indicates that 14-3-3 proteins could play a role in consolidating and strengthening the effects of phosphorylation on TPH2 and that they may be important for the regulation of serotonin function in the nervous system.

scholarly journals Human haem oxygenase-1 induction by nitro-linoleic acid is mediated by cAMP, AP-1 and E-box response element interactions

2009 ◽  
Vol 422 (2) ◽  
pp. 353-361 ◽  
Author(s):  
Marcienne M. WRIGHT ◽  
Junghyun KIM ◽  
Thomas D. HOCK ◽  
Norbert LEITINGER ◽  
Bruce A. FREEMAN ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Site Directed Mutagenesis ◽  
Related Factor ◽  
Intact Cells ◽  
Response Element ◽  
Hek 293 ◽  
293 Cells ◽  
E Box ◽  
Haem Oxygenase ◽  
Anti Inflammatory

Nitro-fatty acid products of oxidative inflammatory reactions mediate anti-inflammatory cell signalling responses. LNO2 (nitrolinoleic acid) induces expression of HO-1 (haem oxygenase-1), an enzyme that catabolizes haem into products exhibiting potent anti-inflammatory properties. In the present manuscript, the molecular mechanisms underlying HO-1 induction by LNO2 were examined in HAEC (human aortic endothelial cells), HEK-293 (human embryonic kidney 293) cells, and in transcription factor-deficient MEF (mouse embryonic fibroblasts). LNO2 induced HO-1 expression in Nrf2 [NF-E2 (nuclear factor-erythroid 2)-related factor 2]-deficient MEF and in HEK-293 cells transfected with Nrf2-specific shRNA (small-hairpin RNA), supporting the fact that LNO2-mediated HO-1 induction can be regulated by Nrf2-independent mechanisms. LNO2 activated expression of a −4.5 kb human HO-1 promoter construct, whereas a −4.0 kb construct with deletion of 500 bp from the 5′ region was unresponsive. Site-directed mutagenesis of a CRE (cAMP-response element) or of a downstream NF-E2/AP-1 (activating protein-1) element, individually, within this 500 bp region modestly reduced activation of the HO-1 promoter by LNO2. Mutations of both the CRE and the NF-E2/AP-1 site also attenuated LNO2-mediated HO-1 promoter expression, whereas the addition of a third mutation in the proximal E-box sequence completely abolished LNO2-induced HO-1 expression. Chromatin immunoprecipitation assays confirmed CREB (CRE-binding protein)-1 binding to the CRE (located at −4.0 kb) and E-box regions (located at −44 bp) of the human HO-1 promoter. A 3C (Chromosome Conformation Capture) assay of intact cells showed LNO2-induced interactions between the CRE- and E-box- containing regions. These observations indicate that regulation of human HO-1 expression by LNO2 requires synergy between CRE, AP-1 and E-box sequences and involves the participation of CREB-1.

scholarly journals A Novel Isoform of Human LZIP Negatively Regulates the Transactivation of the Glucocorticoid Receptor

2009 ◽  
Vol 23 (11) ◽  
pp. 1746-1757 ◽  
Author(s):  
Hyereen Kang ◽  
Yoon Suk Kim ◽  
Jesang Ko
Keyword(s):  
Amino Acids ◽  
Glucocorticoid Receptor ◽  
Transcriptional Activation ◽  
Leucine Zipper ◽  
Target Genes ◽  
Transmembrane Domain ◽  
Negative Regulator ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Basic Leucine Zipper

Abstract The human leucine zipper protein (LZIP) is a basic leucine zipper transcription factor that is involved in leukocyte migration, tumor suppression, and endoplasmic reticulum stress-associated protein degradation. Although evidence suggests a diversity of roles for LZIP, its function is not fully understood, and the subcellular localization of LZIP is still controversial. We identified a novel isoform of LZIP and characterized its function in ligand-induced transactivation of the glucocorticoid receptor (GR) in COS-7 and HeLa cells. A novel isoform of human LZIP designated as “sLZIP” contains a deleted putative transmembrane domain (amino acids 229–245) of LZIP and consists of 345 amino acids. LZIP and sLZIP were ubiquitously expressed in a variety of cell lines and tissues, with LZIP being much more common. sLZIP was mainly localized in the nucleus, whereas LZIP was located in the cytoplasm. Unlike LZIP, sLZIP was not involved in the chemokine-mediated signal pathway. sLZIP recruited histone deacetylases (HDACs) to the promoter region of the mouse mammary tumor virus luciferase reporter gene and enhanced the activities of HDACs, resulting in suppression of expression of the GR target genes. Our findings suggest that sLZIP functions as a negative regulator in glucocorticoid-induced transcriptional activation of GR by recruitment and activation of HDACs.

scholarly journals Distinct Glucocorticoid Receptor Transcriptional Regulatory Surfaces Mediate the Cytotoxic and Cytostatic Effects of Glucocorticoids

1999 ◽  
Vol 19 (7) ◽  
pp. 5036-5049 ◽  
Author(s):  
Inez Rogatsky ◽  
Adam B. Hittelman ◽  
David Pearce ◽  
Michael J. Garabedian
Keyword(s):  
Glucocorticoid Receptor ◽  
Transcriptional Activation ◽  
Molecular Mechanisms ◽  
Target Genes ◽  
Cellular Proliferation ◽  
Antiproliferative Effect ◽  
Glucocorticoid Treatment ◽  
Transcriptional Regulatory ◽  
Human Osteosarcoma Cells ◽  
Transcriptional Regulatory Mechanisms

ABSTRACT Glucocorticoids act through the glucocorticoid receptor (GR), which can function as a transcriptional activator or repressor, to elicit cytostatic and cytotoxic effects in a variety of cells. The molecular mechanisms regulating these events and the target genes affected by the activated receptor remain largely undefined. Using cultured human osteosarcoma cells as a model for the GR antiproliferative effect, we demonstrate that in U20S cells, GR activation leads to irreversible growth inhibition, apoptosis, and repression of Bcl2. This cytotoxic effect is mediated by GR’s transcriptional repression function, since transactivation-deficient mutants and ligands still bring about apoptosis and Bcl2 down-regulation. In contrast, the antiproliferative effect of GR in SAOS2 cells is reversible, does not result in apoptosis or repression of Bcl2, and is a function of the receptor’s ability to stimulate transcription. Thus, the cytotoxic versus cytostatic outcome of glucocorticoid treatment is cell context dependent. Interestingly, the cytostatic effect of glucocorticoids in SAOS2 cells involves multiple GR activation surfaces. GR mutants and ligands that disrupt individual transcriptional activation functions (activation function 1 [AF-1] and AF-2) or receptor dimerization fail to fully inhibit cellular proliferation and, remarkably, discriminate between the targets of GR’s cytostatic action, the cyclin-dependent kinase inhibitors p21Cip1 and p27Kip1. Induction of p21Cip1 is agonist dependent and requires AF-2 but not AF-1 or GR dimerization. In contrast, induction of p27Kip1 is agonist independent, does not require AF-2 or AF-1, but depends on GR dimerization. Our findings indicate that multiple GR transcriptional regulatory mechanisms that employ distinct receptor surfaces are used to evoke either the cytostatic or cytotoxic response to glucocorticoids.

scholarly journals Human and Quail Aromatase Activity Is Rapidly and Reversibly Inhibited by Phosphorylating Conditions

Endocrinology ◽  
2011 ◽  
Vol 152 (11) ◽  
pp. 4199-4210 ◽  
Author(s):  
Thierry D. Charlier ◽  
Nobuhiro Harada ◽  
Jacques Balthazart ◽  
Charlotte A. Cornil
Keyword(s):  
Enzymatic Activity ◽  
Bioinformatic Analysis ◽  
Site Directed Mutagenesis ◽  
General Mechanism ◽  
Aromatase Activity ◽  
Potential Implication ◽  
Hek 293 ◽  
Calcium Dependent ◽  
293 Cells ◽  
Rapid Changes

Besides their slow genomic actions, estrogens also induce rapid physiological responses. To be functionally relevant, these effects must be associated with rapid changes in local concentrations of estrogens. Rapid changes in aromatase activity (AA) controlled by calcium-dependent phosphorylations of the enzyme can alter in a rapid manner local estrogen concentrations, but so far this mechanism was identified only in the avian (quail) brain. We show here that AA is also rapidly down-regulated by phosphorylating conditions in quail ovary homogenates and in various cell lines transfected with human aromatase (HEK 293, Neuro2A, and C6). Enzymatic activity was also rapidly inhibited after depolarization of aromatase-expressing HEK 293 cells with 100 mm KCl, and activity was fully restored when cells returned to control conditions. Western blot analysis demonstrated that the reduction of enzymatic activity is not due to protein degradation. We next investigated by site-directed mutagenesis the potential implication in the control of AA of specific aromatase residues identified by bioinformatic analysis. Mutation of the amino acids S118, S247, S267, T462, T493, or S497 to alanine, alone or in combination, did not block the rapid inhibition of enzymatic activity induced by phosphorylating conditions, but basal AA was markedly decreased in the S118A mutant. Altogether, these results demonstrate that the rapid inhibition of AA is a widespread and fully reversible process and that phosphorylation of specific residues modulate AA. These processes provide a new general mechanism by which local estrogen concentration can be rapidly altered in the brain and other tissues.

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