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T Decker

Publications and source records attributed to T Decker.

At least 37 records · Page 2Linked to original sources
PubMed1995

Colony-stimulating factors and interferon-gamma activate a protein related to MGF-Stat 5 to cause formation of the differentiation-induced factor in myeloid cells.

The Jak-Stat pathway of intracellular signals is used by growth factor- and cytokine receptors to induce gene transcription. We have recently reported that differentiation of myeloid cells, induced by phorbol ester, interferon-gamma (IFN-gamma) or colony-stimulating factor-1 (CSF-1) is accompanied by the activation of the differentiation-induced factor (DIF). Activated DIF specifically associates with a subclass of gamma-interferon activation site (GAS)-like DNA elements. We now report that GM-CSF, which like CSF-1 promotes the generation of mature macrophages, activates DIF. No activation was observed after treatment with the granulocyte growth and differentiation factor G-CSF. Antibodies raised against a Stat family protein, designated mammary gland factor-Stat 5 (MGF-Stat 5), reacted with DIF induced by either CSF-1, GM-CSF or IFN-gamma. Antisera to other known Stats were without effect on the DIF complex in electrophoretic mobility shift assays (EMSA). A 112 kDa protein could be isolated from either GM-CSF- or IFN-gamma-treated cells by GAS oligonucleotide precipitation. This protein reacted with antibodies to both MGF-Stat 5 and phosphotyrosine. MGF-Stat 5 and closely related proteins thus define a subfamily of Stat transcription factors that are present in a variety of cell types and are required for the onset of immediate gene expression in response to differentiating stimuli.

Blotting, Western↗
PubMed1995

Activity of Stat family transcription factors is developmentally controlled in cells of the macrophage lineage.

Stat family transcription factors are activated in response to a variety of cytokines to bind to a class of DNA elements termed gamma interferon activation site (GAS)-like elements. Here we investigate two GAS-binding transcription factors, the gamma-interferon activation factor (GAF) and the differentiation-induced factor (DIF) that are activated by interferon-gamma (IFN-gamma) in U937 cells. Treatment of U937 cells with phorbol ester (TPA) induces differentiation from a promonocyte into a monocyte stage of macrophage development. Monocytic differentiation led to an increased transcriptional response of GAS-containing genes to IFN-gamma. TPA treatment also caused a profound change in the IFN-gamma activation of GAF and DIF. GAF DNA-binding activity was activated much better in the monocyte stage and the GAF constituent Stat 1 showed increased phosphorylation. In contrast, DIF activation by IFN-gamma was found in promonocytes but was virtually absent in monocytes. Moreover, DIF activation was observed during TPA-induced monocytic differentiation and after treatment of macrophages with the macrophage differentiation factor CSF-1. Our data suggest DIF to be part of a developmental program leading to terminal macrophage differentiation and GAF to be a transcription factor bringing about the stronger activation response of mature macrophages to IFN-gamma.

Cell Differentiation↗
PubMed1995

Interferon regulatory factor 1 is required for mouse Gbp gene activation by gamma interferon.

Full-scale transcriptional activation of the mouse Gbp genes by gamma interferon (IFN-gamma) requires protein synthesis in embryonic fibroblasts. Although the Gbp-1 and Gbp-2 promoters contain binding sites for transcription factors Stat1 and IFN regulatory factor 1 (IRF-1), deletion analysis revealed that the Stat1 binding site is dispensable for IFN-gamma inducibility of Gbp promoter constructs in transfected fibroblasts. However, activation of the mouse Gbp promoter by IFN-gamma requires transcription factor IRF-1. Transient overexpression of IRF-1 cDNA in mouse fibroblasts resulted in high-level expression of Gbp promoter constructs. Unlike wild-type cells, IRF-1% embryonic stem cells lacking functional transcription factor IRF-1 contained very low levels of Gbp transcripts that were not increased in response to differentiation or treatment with IFN-gamma. Treatment of IRF-1% mice with IFN-gamma resulted in barely detectable levels of Gbp RNA in spleens, lungs, and livers, whereas such treatment induced high levels of Gbp RNA in the organs of wild-type mice. These observations suggest two alternative pathways for transcriptional induction of genes in response to IFN-gamma: immediate response that results from activation of preformed Stat1 and delayed response that results from induced de novo synthesis of transcription factor IRF-1.

3T3 Cells↗
PubMed1995

Differentiation-regulated serine phosphorylation of STAT1 promotes GAF activation in macrophages.

Gamma interferon (IFN-gamma), a macrophage-activating cytokine, modulates gene expression through the activity of a transcription factor designated IFN-gamma activation factor (GAF). GAF is formed after phosphorylation on tyrosine and dimerization of the 91-kDa protein STAT1. We have recently reported that differentiation of the promonocytic cell line U937 into monocytes increases the amount of cellular GAF after IFN-gamma treatment and at the same time increases the phosphorylation of STAT1. Here we show that activation of the JAK family kinases, which are instrumental in mediating STAT1 phosphorylation on tyrosine, did not increase upon monocytic U937 differentiation. Consistent with this finding, levels of STAT1 tyrosine phosphorylation were virtually identical in promonocytic and monocytic U937 cells. Analysis of STAT1 phosphoamino acids and mapping of phosphopeptides showed an IFN-gamma-dependent increase in Ser phosphorylation in differentiated cells. Analyses of STAT1 isoforms by two-dimensional gel electrophoresis demonstrated a differentiation-induced shift toward more acidic isoforms. All isoforms were equally sensitive to subsequent tyrosine phosphorylation, as indicated by a sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobility shift typical for tyrosine-phosphorylated STAT1. Consistent with the importance of Ser phosphorylation for high-affinity binding to the IFN-gamma activation site sequence, phosphatase 2A treatment strongly reduced the formation of IFN-gamma activation site-GAF complexes in an electrophoretic mobility shift assay. Our data indicate that the activity of GAF is modulated by STAT1 serine kinases/phosphatases and suggest that this mechanism is employed in the developmental control of macrophage responsiveness to IFN-gamma.

Amino Acids↗
PubMed1994

The interferon-inducible GBP1 gene: structure and mapping to human chromosome 1.

We have isolated DNA clones containing the interferon (IFN)-inducible guanylate-binding protein-1-encoding gene (GBP1) from a human genomic library. Two overlapping phage clones contained the entire GBP1 gene. The 2880 bp corresponding to the GBP1 cDNA were subdivided into eleven exons which were interrupted by a total of 9500 bp of intron DNA. All exon/intron junctions contained consensus splice donor and acceptor sequences. Using hybrid rodent cell lines containing human chromosomes, GBP1 was mapped to human chromosome 1. In addition to GBP1, we detected and partially characterized a novel gene, GBP3, with a structure related to GBP1 and a high degree of sequence homology to both GBP1 and GBP2. Our data suggest that the GBP family of genes contains members other than the previously characterized GBP1 and GBP2.

Amino Acid Sequence↗
PubMed1994

IL-10-induced factors belonging to the p91 family of proteins bind to IFN-gamma-responsive promoter elements.

Expression of the gene encoding the high affinity IgG receptor (Fc gamma RI) is stimulated by IFN-gamma through a promoter element designated gamma-IFN activation site (GAS). This sequence binds a transcription factor designated gamma-IFN activation factor (GAF). GAF-GAS complexes contain an IFN-regulated 91-kDa protein (p91). In mouse peritoneal macrophages, IL-4 and IL-10 influenced both basal and IFN-gamma-induced expression of Fc gamma RI in opposite ways: IL-10 was stimulatory and IL-4 repressed Fc gamma RI expression. IL-4 or IL-10 did not affect the activation of GAF by IFN-gamma, but both activated the binding of latent, receptor-activated factors (RAFTs) to the Fc gamma RI GAS. RAFTs-IL-4 and -IL-10 migrated similarly in electrophoretic mobility shift assays but could be distinguished through their specificities for different GAS sequences and their reactivity with anti-p91 antisera. These experiments also revealed two distinct RAFTs-IL-10 to be members of the p91 family of proteins. The data suggest GAS-related elements to integrate signals from IFN-gamma-, IL-4- and IL-10-activated signaling paths.

Animals↗
PubMed1994

A novel interferon-alpha-regulated, DNA-binding protein participates in the regulation of the IFP53/tryptophanyl-tRNA synthetase gene.

We have investigated the transcriptional response of the IFP53/tryptophanyl-tRNA synthetase gene to interferon-alpha (IFN-alpha). A single gamma-interferon activation site (GAS) in proximity to the transcription start sites was found to mediate the response of the IFP53 gene to IFN-alpha. This DNA element bound two distinct protein factors, alpha-interferon activation factor 1 (AAF1) and AAF2, which were rapidly activated in the cytoplasm of IFN-alpha-treated HeLa cells. AAF1, like the gamma-interferon activation factor, bound to the GAS from different IFN-responsive promoters and contained the 91-kDa ISGF3 protein (p91). However, in complexes with the IFP53 or Ly6A/E GAS, p91 was the only ISGF3 protein, whereas in the case of the GBP GAS, the 48-kDa protein (p48) was also present. AAF2 was found to preferentially bind to the IFP53 GAS, but not at all to the GBP GAS, and contained no ISGF3 protein. Therefore, GAS-binding regulatory factors in the IFN-alpha response can either consist of proteins found in ISGF3 or be formed by distinct proteins that are similarly linked to IFN-alpha-induced signal transduction.

Blotting, Western↗
PubMed1994

Cytokines and growth factors signal through tyrosine phosphorylation of a family of related transcription factors.

The ability of cytokines to activate distinct but overlapping sets of genes defines their characteristic biological response. We now show that IFN gamma, IL-3, IL-4, IL-6, erythropoietin, EGF, and CSF-1 activate differing members of a family of latent cytoplasmic transcription factors. Although these factors have distinct physical and functional properties and exhibit different patterns of expression, they share many important features, including recognition of a related set of enhancer elements, rapid activation, tyrosine phosphorylation, and cross-reactivity to antibodies against p91, a cytoplasmic signaling protein activated by IFN alpha, IFN gamma, and IL-6. These shared features point to either parallel or common patterns of signal transduction. A general model of cytokine signal transduction is presented, in which receptor-associated tyrosine kinases activate ligand-specific members of a family of signal-transducing factors. Once activated, these factors carry their signals to the nucleus, where they bind a family of related enhancer elements.

Base Sequence↗
PubMed1994

A factor induced by differentiation signals in cells of the macrophage lineage binds to the gamma interferon activation site.

Rapid transcriptional induction of genes in response to gamma interferon (IFN-gamma) is mediated by the IFN-gamma activation site (GAS) and its cognate protein, the IFN-gamma activation factor (GAF). We describe a GAS-associated, differentiation-induced factor (DIF) as a potential molecular link between the activities of IFN-gamma and of growth and differentiation factors. DIF DNA binding was activated by colony-stimulating factor 1 in murine macrophages and also during tetradecanoyl phorbol acetate-induced differentiation or IFN-gamma treatment in myeloid U937 cells. IFN-gamma activation of DIF decreased significantly upon monocytic differentiation. DIF binding to DNA was inhibited by antiphosphotyrosine antibodies and could be induced by treatment of U937 cells with vanadate. Unlike GAF, DIF-DNA complexes did not contain the 91-kDa protein (p91) from ISGF-3. DIF bound with high affinity to GAS from the promoters of the IFP 53/tryptophanyl-tRNA synthetase and Fc gamma RI genes, intermediate affinity to the Ly6A/E GAS, and low affinity to the guanylate-binding protein GAS. DIF may belong to a family of cytokine- or growth factor-induced factors binding with variable affinities to GAS-related elements: the interleukin-6-responsive acute-phase response factor associated with GAS from different IFN-inducible promoters but with a different preference of binding compared with DIF. The sis-inducible element of the c-fos promoter bound GAF but not DIF. However, the sis-inducible element could be changed by point mutation to compete for GAF and DIF binding. Our data show DIF to be a novel DNA-binding protein which is activated in response to differentiating signals. Moreover, they suggest that a family of cytokine- or growth factor-regulated proteins integrates and coordinates the responses to cytokines and to growth and differentiation factors by binding to GAS-related elements.

Animals↗
PubMed1994

The signalling pathways of interleukin-6 and gamma interferon converge by the activation of different transcription factors which bind to common responsive DNA elements.

Interleukin-6 (IL-6) and gamma interferon (IFN-gamma) induce a partially overlapping set of genes, including the genes for interferon regulatory factor 1 (IRF-1), intercellular adhesion molecule 1 (ICAM-1), and the acute-phase protein alpha 2-macroglobulin. We report here that the rat alpha 2-macroglobulin promoter is activated by IFN-gamma in human hepatoma (HepG2) cells and that the IFN-gamma response element maps to the same site previously defined as the acute-phase response element (APRE), which binds the IL-6-activated transcription factor APRF (acute-phase response factor). As was reported for fibroblasts, the IFN-gamma-regulated transcription factor GAF is phosphorylated at tyrosine after IFN-gamma treatment of HepG2 cells. IFN-gamma posttranslationally activates a protein which specifically binds to the alpha 2-macroglobulin APRE. This protein is shown to be identical or closely related to GAF. Although APRF and GAF are shown to represent different proteins, their binding sequence specificities are very similar. APRF and GAF bind equally well to the APRE sequences of various acute-phase protein genes as well as to the IFN-gamma response elements of the IRF-1, ICAM-1, and other IFN-gamma-inducible genes. Transient transfection analysis revealed that the IFN-gamma response elements of the IRF-1 and ICAM-1 promoters are able to confer responsiveness to both IFN-gamma and IL-6 onto a heterologous promoter. Therefore, APRF and GAF are likely to be involved in the transcriptional induction of these immediate-early genes by IL-6 and IFN-gamma, respectively. Taken together, these results demonstrate that two functionally distinct hormones, IL-6 and IFN-gamma, act through common regulatory elements to which different transcription factors sharing almost the same sequence specificity bind.

Alkaloids↗
PubMed1994

Coordinate expression of the lineage-specific growth factor colony-stimulating factor (CSF)-1 and its receptor selectively promotes macrophage maturation during differentiation of multipotential progenitor cells.

The multipotent hematopoietic precursor line A4GMV#2, derived by infection of FDCP-mix cells with a retroviral vector expressing the granulocyte-macrophage colony-stimulating factor (CSF) gene, proliferates continuously in interleukin 3 and presents the unique advantage of synchronous granulocyte and macrophage differentiation upon interleukin 3 withdrawal. Using this system, we showed previously that the mRNAs for lineage-specific receptors (granulocyte-CSF receptors, CSF-1 receptors, and Erythropoietin receptors) and ligands (granulocyte-CSF and CSF-1) are up-regulated during myeloid maturation. Here we address the specific question of the regulation of the expression of CSF-1 and its receptor and of their relevance to macrophage differentiation. Both genes were transcribed with equal efficiency in undifferentiated and differentiating cells. CSF-1 mRNA was detected in undifferentiated cells and increased slightly in the early phases of differentiation. CSF-1 receptor mRNA, absent in undifferentiated cells, accumulated early in differentiation (24 h) and remained constant thereafter. The production of both proteins, detected later during the differentiation of A4GMV#2 cells and of bone marrow-derived myeloid precursors, was therefore controlled at the posttranscriptional level. CSF-1 was produced by cells of the macrophage lineage and accumulated in mature phagocytes. A neutralizing anti-CSF-1 serum selectively impaired macrophage differentiation of A4GMV#2 cells and, most significantly, of primary myeloid precursors. These data indicate that CSF-1 and its receptor interact productively during differentiation and that the resulting autocrine stimulation selectively promotes macrophage maturation.

Amino Acid Sequence↗
PubMed1993

The gene encoding IFP 53/tryptophanyl-tRNA synthetase is regulated by the gamma-interferon activation factor.

We have obtained genomic DNA encoding the interferon-gamma (IFN-gamma)-inducible IFP 53/tryptophanyl-tRNA synthetase. Comparison with several different IFP 53 cDNA clones revealed a complex pattern of alternatively spliced 5'-untranslated regions. The interferon-responsive region within the IFP 53 promoter was found to contain a gamma-interferon activation site (GAS) but not the interferon-stimulated response element and to bind the gamma-interferon activation factor (GAF). GAF.GAS complexes contained the IFN-regulated 91-kDa protein. Competition experiments defined the GAS boundaries and showed that GAF binding to the IFP 53 GAS could be prevented by an excess of the IFN-gamma response regions of several other IFN-gamma-inducible genes. We thus provide evidence for a central role of GAS.GAF in gene transcription mediated by IFN-gamma and suggest a consensus sequence defining more precisely the requirements for GAF binding to DNA.

Base Sequence↗
PubMed1993

The response of gamma interferon activation factor is under developmental control in cells of the macrophage lineage.

Gamma interferon activation factor (GAF) rapidly induces transcriptional activation of gamma interferon (IFN-gamma)-responsive genes. Conversion of the GAF from a latent cytoplasmic to an activated, DNA-binding form is an immediate step in the cellular response to IFN-gamma. The amount of IFN-gamma-activated GAF, measured by exonuclease III protection or gel shift assays, increased strongly upon monocytic differentiation of U937 cells. Activated GAF contained the IFN-responsive 91-kDa protein as its DNA-binding activity in gel shift or exonuclease III assays could be inhibited through direct addition of specific antiserum, and it was not present in p91-immunodepleted extracts. There was a differentiation-induced increase in the amount of nonphosphorylated (latent) p91. Transcription rate measurement demonstrated a strong induction of the p91 gene during monocytic differentiation of U937 cells. The amount of p91 which was rapidly phosphorylated in response to IFN-gamma was found to be much higher in the differentiated cells and suggested a differentiation-controlled increase in the signaling leading to p91 phosphorylation. Concomitantly with a better GAF response, transcriptional activation of IFN-gamma-induced genes and the expression of GAF-dependent, transfected reporter plasmids increased in differentiated U937 monocytes. The promonocyte-monocyte transition also affected the IFN-alpha-responsive transcription factor ISGF-3. Differentiated U937 cells contained more of both the alpha-component p91 and the gamma-component p48, which constitutes the DNA-binding subunit of the complex. Our study thus provides evidence that the synthesis of specific transcription factors can be a regulated event to control the cytokine responsiveness of cells during development.

Binding Sites↗
PubMed1992

Transcriptional induction of IFN-gamma-responsive genes is modulated by DNA surrounding the interferon stimulation response element.

The 9/27 and GBP mRNAs are both inducible by Interferon-gamma (IFN-gamma). The promoters of both genes contain an Interferon Stimulation Response Element (ISRE), but while the GBP gene is strongly induced transcriptionally by IFN-gamma the response of the 9/27 promoter is very weak. We investigated the molecular basis for this difference. The different IFN-gamma-responsiveness was found to have more than one reason. First, 9/27 promoter DNA was unable to bind the Gamma Interferon Activation Factor (GAF) with a single high affinity site. It efficiently competed for the association of the GAF with the GBP promoter but this competition was due to the presence of two low affinity sites, the ISRE and an ISRE-like sequence, suggesting that the GAS and ISRE, though both having clear preferences for specific proteins, may nevertheless share a certain degree of structural homology. Second, the 9/27 and GBP ISREs differed markedly in their affinities for regulatory proteins (ISGFs 1,2,3) and the GBP ISRE was more potent in mediating IFN-gamma-induced promoter activity in transient transfection. Third and most importantly, however, the strong difference between the IFN-gamma response of the two promoters was mainly due to the sequences surrounding the ISRE: the positive-acting GAS on one side and sequences with silencing properties 5' and 3' of the 9/27 ISRE on the other side. The data thus show mechanisms to both up- and down-regulate the activity of the ISRE.

Base Sequence↗
PubMed1992

Interferon-gamma regulates expression of a novel keratin class I gene.

Interferon (IFN)-gamma has been implicated in the pathogenesis of several autoimmune disorders and inflammatory skin diseases. To identify novel mediators involved in the IFN-gamma response we have used differential hybridization of a cDNA library prepared from IFN-gamma-treated HeLa cells to isolate a gene that is induced following treatment with IFN-gamma. We report here the molecular cloning and characterization of a cDNA detecting a 1.6-kb mRNA that accumulated in response to IFN-gamma but not in response to IFN-alpha or IFN-beta. The gene is regulated by IFN-gamma in human cell lines of epithelial origin. The mRNA encodes a predicted protein of 432 amino acids and the primary structure of the protein demonstrates that it is a novel member of developmentally regulated keratin class I genes.

Amino Acid Sequence↗
PubMed1992

Double-stranded RNA and interferon-alpha induce transcription through different molecular mechanisms.

Double-stranded (ds) RNA stimulates the synthesis of several mRNAs known to be induced by type I interferons (IFNs). In this report, it is shown that the IFN-alpha stimulated genes (ISGs) 15, 54, 56, and GBP are transcriptionally induced by dsRNA. Transcriptional stimulation occurred in the presence of the protein synthesis inhibitor cycloheximide (CHX), indicating that inducibility was directly mediated by dsRNA through the action of preformed proteins. ISGF-3, the protein complex mediating primary transcriptional induction of ISGs by IFN-alpha, was not activated by dsRNA in the presence of CHX. Additionally, DNA-binding activity of ISGF-2/IRF-1, a protein involved in the regulation of the IFN-beta gene and ISGs, did not correlate with dsRNA-induced transcriptional induction of ISGs. This suggests that dsRNA and IFN-alpha induce ISGs through different molecular mechanisms.

Cycloheximide↗
PubMed1992

Interferon induction of gene transcription analyzed by in vivo footprinting.

The promoters of two interferon-induced genes (the ISG54 and guanylate-binding protein [GBP] genes) have been analyzed in whole cells and in isolated nuclei by using a new genomic sequencing technique. The ISG54 gene contains an interferon-simulating response element (ISRE), earlier shown to be necessary and sufficient for alpha interferon (IFN-alpha) induction, that appeared complexed with proteins in both transcribing and nontranscribing cells. However, the extent of protection and hypersensitivity to DNase I or dimethyl sulfate within the ISRE region was changed upon transcriptional induction, suggesting the binding of different factors in different transcriptional states. In addition to the ISRE, the GBP gene needs a newly recognized DNA element, called the GAS, that partly overlaps the ISRE for full induction by either IFN-alpha or IFN-gamma. This GAS element was transiently protected against DNase I in the nuclei of interferon-treated cells but was not protected at later times when transcription reached maximal levels. Thus, the GAS-binding activity may be necessary only transiently for the initial assembly of a transcription initiation complex on the GBP promoter. Dimethyl sulfate methylation of genomic DNA performed on intact cells showed a characteristic sensitivity over the GAS that correlated with transcription levels and that persisted longer than did DNase I protection over the GAS. These results demonstrate the involvement of the GAS in IFN-alpha and -gamma induction of GBP and suggest the presence of an altered DNA conformation or a small protein in the major groove of the GAS associated with ongoing GBP transcription.

Base Sequence↗