In vitro activation of the transcription factor gamma interferon activation factor by gamma interferon: evidence for a tyrosine phosphatase/kinase signaling cascade

1993 ◽  
Vol 13 (3) ◽  
pp. 1634-1640
Author(s):  
K Igarashi ◽  
M David ◽  
D S Finbloom ◽  
A C Larner
Keyword(s):  
Tyrosine Phosphatase ◽  
Gamma Interferon ◽  
Cell Surface Receptor ◽  
Signaling Cascade ◽  
Kinase Signaling ◽  
Gamma Activation ◽  
Ifn Gamma ◽  
Genistein Treatment ◽  
Activation Factor

Although it has been well documented that the biological activities of gamma interferon (IFN-gamma) are initiated through interaction with its cell surface receptor, the signal transduction mechanisms which mediate the effects of this cytokine have remained unclear. In order to facilitate a better understanding of IFN-gamma signaling, we have designed an assay using human fibroblast cell homogenates in which IFN-gamma activates the formation of the IFN-gamma activation factor (GAF) transcription complex. GAF mediates the rapid transcriptional activation of the guanylate-binding protein gene by IFN-gamma. Activation of GAF in homogenates required ATP, but not Ca2+ or GTP. Fractionation of homogenates indicated that both the pellet (18,000 x g) and the remaining cytoplasmic fraction were required for GAF activation by IFN-gamma. In intact cells and cell homogenates, the activation of GAF was prevented by the specific tyrosine kinase inhibitor genistein. Treatment of GAF-containing nuclear extracts with either monoclonal antiphosphotyrosine antibody or protein tyrosine phosphatase prevented the assembly of the transcription complex, indicating that its formation required phosphorylation of tyrosine residues. Furthermore, the tyrosine phosphatase inhibitors phenylarsine oxide and zinc chloride also inhibited GAF formation in vitro, but only if these agents were added to cell homogenates before IFN-gamma was added. The addition of either agent 5 min after IFN-gamma had no effect. These results provide the first evidence for an IFN-gamma-regulated tyrosine phosphatase/kinase signaling cascade that permits this cytokine to activate the transcription of an early-response gene.

scholarly journals In vitro activation of the transcription factor gamma interferon activation factor by gamma interferon: evidence for a tyrosine phosphatase/kinase signaling cascade.

1993 ◽  
Vol 13 (3) ◽  
pp. 1634-1640 ◽  
Author(s):  
K Igarashi ◽  
M David ◽  
D S Finbloom ◽  
A C Larner
Keyword(s):  
Tyrosine Phosphatase ◽  
Gamma Interferon ◽  
Cell Surface Receptor ◽  
Signaling Cascade ◽  
Kinase Signaling ◽  
Gamma Activation ◽  
Ifn Gamma ◽  
Genistein Treatment ◽  
Activation Factor

Although it has been well documented that the biological activities of gamma interferon (IFN-gamma) are initiated through interaction with its cell surface receptor, the signal transduction mechanisms which mediate the effects of this cytokine have remained unclear. In order to facilitate a better understanding of IFN-gamma signaling, we have designed an assay using human fibroblast cell homogenates in which IFN-gamma activates the formation of the IFN-gamma activation factor (GAF) transcription complex. GAF mediates the rapid transcriptional activation of the guanylate-binding protein gene by IFN-gamma. Activation of GAF in homogenates required ATP, but not Ca2+ or GTP. Fractionation of homogenates indicated that both the pellet (18,000 x g) and the remaining cytoplasmic fraction were required for GAF activation by IFN-gamma. In intact cells and cell homogenates, the activation of GAF was prevented by the specific tyrosine kinase inhibitor genistein. Treatment of GAF-containing nuclear extracts with either monoclonal antiphosphotyrosine antibody or protein tyrosine phosphatase prevented the assembly of the transcription complex, indicating that its formation required phosphorylation of tyrosine residues. Furthermore, the tyrosine phosphatase inhibitors phenylarsine oxide and zinc chloride also inhibited GAF formation in vitro, but only if these agents were added to cell homogenates before IFN-gamma was added. The addition of either agent 5 min after IFN-gamma had no effect. These results provide the first evidence for an IFN-gamma-regulated tyrosine phosphatase/kinase signaling cascade that permits this cytokine to activate the transcription of an early-response gene.

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

1994 ◽  
Vol 14 (2) ◽  
pp. 1364-1373 ◽  
Author(s):  
A Eilers ◽  
M Baccarini ◽  
F Horn ◽  
R A Hipskind ◽  
C Schindler ◽  
...  
Keyword(s):  
Growth Factor ◽  
Dna Binding ◽  
Binding Protein ◽  
Trna Synthetase ◽  
Gamma Interferon ◽  
U937 Cells ◽  
Gamma Activation ◽  
Ifn Gamma ◽  
Differentiation Factors ◽  
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.

scholarly journals Ligand-dependent and -independent activation of the transcription factor γRF-1 in a cell-free system

1995 ◽  
Vol 310 (2) ◽  
pp. 461-467 ◽  
Author(s):  
C A Feghali ◽  
T M Wright
Keyword(s):  
Transcription Factor ◽  
Tyrosine Phosphatase ◽  
Cell Fractionation ◽  
Dependent Manner ◽  
Sodium Orthovanadate ◽  
Ifn Gamma ◽  
Free System ◽  
Cell Free System ◽  
A Cell

gamma RF-1 is a recently identified transcription factor induced by interferon-gamma (IFN-gamma) which binds to a unique palindromic enhancer, gamma RE-1, in the promoter of the mig gene. This paper describes the ligand-dependent and ligand-independent activation of gamma RF-1 in a cell-free system. gamma RF-1 activity was induced by IFN-gamma in a time-dependent manner from 5 to 60 min in lysates prepared from the human monocytic leukaemia line THP-1 and the human epidermoid carcinoma line A431. The activation of gamma RF-1 in vitro required both ATP and an inhibitor of tyrosine phosphatases (sodium orthovanadate or pervanadate). In the presence of limiting concentrations (micromolar) of ATP, activation was also dependent upon stimulation with IFN-gamma, whereas at millimolar concentrations of ATP, gamma RF-1 was activated by either sodium orthovanadate or pervanadate in the absence of ligand. Based on cell fractionation studies, both membrane and cytosol components were essential for activation of gamma RF-1 in vitro. Consistent with a role for one or more tyrosine kinases in the activation of gamma RF-1, its DNA binding activity was blocked by monoclonal anti-phosphotyrosine antibodies and by the tyrosine kinase inhibitors genistein, lavendustin A and herbimycin A. A comparison with recently described pathways of IFN-mediated transcription factor regulation indicates that the in vitro activation of gamma RF-1 is unique, requiring both membrane and cytosol fractions and inhibition of endogenous tyrosine phosphatase activity.

Two distinct alpha-interferon-dependent signal transduction pathways may contribute to activation of transcription of the guanylate-binding protein gene

1991 ◽  
Vol 11 (10) ◽  
pp. 5147-5153
Author(s):  
T Decker ◽  
D J Lew ◽  
J E Darnell
Keyword(s):  
Binding Protein ◽  
Alpha Interferon ◽  
Gamma Activation ◽  
Gene Encoding ◽  
Ifn Gamma ◽  
Ifn Alpha ◽  
Activation Factor ◽  
Activation Conditions ◽  
Guanylate Binding Protein

The promoter of the gene encoding a cytoplasmic guanylate-binding protein (GBP) contains two overlapping elements: the interferon stimulation response element (ISRE), which mediates alpha interferon (IFN-alpha)-dependent transcription, and the IFN-gamma activation site (GAS), which is required for IFN-gamma-mediated stimulation. The ISRE binds a factor called ISGF-3 that is activated by IFN-alpha but not by IFN-gamma. The GAS binds a protein that is activated by IFN-gamma, which we have termed GAF (IFN-gamma activation factor; T. Decker, D. J. Lew, J. Mirkovitch, and J. E. Darnell, Jr., EMBO J., in press; D. J. Lew, T. Decker, I. Strehlow, and J. E. Darnell, Jr., Mol. Cell. Biol. 11:182-191, 1991). We now find that the GAS is also an IFN-alpha-responsive element in vivo and that IFN-alpha (in addition to activating ISGF-3) rapidly activates a GAS-binding factor, the IFN-alpha activation factor (AAF). The AAF has characteristics very similar to those of the previously described GAF. Through the use of inhibitors of protein synthesis and inhibitors of protein kinases, the activation conditions of AAF, GAF, and ISGF-3 could be distinguished. Therefore, not only do IFN-alpha and IFN-gamma stimulate transcription of GBP through different receptors linked to different signaling molecules, but occupation of the IFN-alpha receptor apparently leads to the rapid activation of two different DNA-binding proteins through the use of different intracellular pathways.

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

1993 ◽  
Vol 13 (6) ◽  
pp. 3245-3254
Author(s):  
A Eilers ◽  
D Seegert ◽  
C Schindler ◽  
M Baccarini ◽  
T Decker
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Cellular Response ◽  
Gamma Interferon ◽  
U937 Cells ◽  
Monocytic Differentiation ◽  
Ifn Gamma ◽  
Exonuclease Iii ◽  
Activation Factor ◽  
Gel Shift

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.

scholarly journals Obligatory role of gamma interferon in T cell-replacing factor-dependent, antigen-specific murine B cell responses.

1985 ◽  
Vol 161 (5) ◽  
pp. 953-971 ◽  
Author(s):  
M Brunswick ◽  
P Lake
Keyword(s):  
T Cell ◽  
B Cell ◽  
Cell Activation ◽  
Human Peripheral Blood ◽  
Gamma Interferon ◽  
Spleen Cells ◽  
Ifn Gamma ◽  
Murine Spleen

The role of gamma interferon (IFN-gamma) in T cell-replacing factor (TRF) activity for antigen-specific plaque-forming cell (PFC) responses in vitro was studied using antibodies to murine IFN-gamma (Mu IFN-gamma). TRF activity was present in supernatants (Sn) of Con A- or mixed leukocyte reaction-stimulated murine spleen cells as well as in an IL-2-rich fraction of phytohemagglutinin-stimulated human peripheral blood lymphocyte Sn and in the Sn of the Gibbon T lymphoma MLA-144. The human TRF was highly active with cells from nu/nu mice and normal mice but not with cells from animals with the xid immunologic defect, similar to the activity of murine TRF. Antibodies to IFN-gamma consisted of hyper-immune rabbit antisera, IFN-gamma affinity-purified rabbit immunoglobulin and an interspecies hybridoma specific for Mu IFN-gamma. The results show that the activities of all preparations of TRF are markedly diminished or abrogated by antibody to Mu IFN-gamma but not by antibodies to human IFN-gamma (Hu IFN-gamma), nor by normal rabbit sera or purified rabbit Ig. The degree of inhibition was dose dependent and was quantitatively reversed by the addition to the cultures of recombinant-derived Mu IFN-gamma (Mu rIFN-gamma) but not Hu rIFN-gamma. This reversal was fully antigen specific and thus not attributable to polyclonal B cell activation by IFN-gamma, which is inactive alone in the TRF assay. Kinetic analysis shows that IFN-gamma must act by 24-48 h to produce PFC responses at 4 d. Together, the data demonstrate that IFN-gamma is a necessary mediator for TRF effects and that IFN-gamma is induced by TRF from T-depleted murine spleen cells in sufficient quantity to support large antibody responses. The source of this IFN-gamma may be the potent natural killer cells that are induced in cultures stimulated with TRF.

scholarly journals In vitro activation of a transcription factor by gamma interferon requires a membrane-associated tyrosine kinase and is mimicked by vanadate.

1993 ◽  
Vol 13 (7) ◽  
pp. 3984-3989 ◽  
Author(s):  
K Igarashi ◽  
M David ◽  
A C Larner ◽  
D S Finbloom
Keyword(s):  
Dna Binding ◽  
Tyrosine Kinase ◽  
Transcriptional Activation ◽  
Human Peripheral Blood ◽  
Protein S ◽  
Early Response ◽  
Gamma Interferon ◽  
Blood Monocytes ◽  
Ifn Gamma

Gamma interferon (IFN-gamma) activates the formation of a DNA-binding protein complex (FcRF gamma) that recognizes the gamma response region (GRR) of the promoter for the human high-affinity Fc gamma receptor. In a membrane-enriched fraction prepared from human peripheral blood monocytes, IFN-gamma activation of FcRF gamma occurred within 1 min and was ATP dependent. Activation of FcRF gamma required a tyrosine kinase activity, and recognition of the GRR sequence by FcRF gamma could be abrogated by treatment with a tyrosine-specific protein phosphatase. Treatment of cells with vanadate alone resulted in the formation of FcRF gamma without the need for IFN-gamma. UV cross-linking and antibody competition experiments demonstrated that the FcRF gamma complex was composed of at least two components: the 91-kDa protein of the IFN-alpha-induced transcription complex ISGF3 and a 43-kDa component that bound directly to the GRR. Therefore, specificity for IFN-induced transcriptional activation of early response genes requires at least two events: (i) ligand-induced activation of membrane-associated protein by tyrosine phosphorylation and (ii) formation of a complex composed of an activated membrane protein(s) and a sequence-specific DNA-binding component.

scholarly journals Phenotypic characterization of gamma interferon-induced human monocyte polykaryons

Blood ◽  
1985 ◽  
Vol 66 (6) ◽  
pp. 1241-1246 ◽  
Author(s):  
JB Weinberg ◽  
MM Hobbs ◽  
MA Misukonis
Keyword(s):  
Acid Phosphatase ◽  
Surface Membrane ◽  
Human Monocyte ◽  
Gamma Interferon ◽  
Cell Protein ◽  
Phenotypic Characterization ◽  
Nonspecific Esterase ◽  
Polystyrene Spheres ◽  
Ifn Gamma

Abstract Multinucleated giant cells of mononuclear phagocyte origin (monocyte or macrophage polykaryons [MPs] ) are seen in numerous different normal and pathologic states. We have previously shown that gamma interferon (IFN-gamma) induces fusion of uninuclear monocytes (UMs) to form MPs. This study was designed to characterize these IFN-gamma-induced MPs. Control and IFN-gamma-treated UMs and MPs did not have peroxidase activity, but they stained intensely for nonspecific esterase and acid phosphatase. The esterase of UMs and MPs was abolished by fluoride, but the acid phosphatase of UMs and MPs was only minimally decreased by tartrate. The phagocytosis of polystyrene spheres and glutaraldehyde- fixed erythrocytes by MPs was moderately depressed as compared with control or treated UMs, whereas the phagocytosis of IgG-coated erythrocytes was markedly depressed. Populations of control monocytes produced less H2O2 in response to 200 nmol/L of phorbol myristate acetate than did IFN-gamma-treated monocytes (37 +/- 7 v 199 +/- 29 nmol/h per milligram of cell protein). However, when examined microscopically, individual MPs had less ability to reduce NBT (18% +/- 5% positive for MP, 91% +/- 3% for treated UMs, and 67% +/- 3% for control UMs). The surface membrane antigens Leu M3, OKM1 (C3bi receptor), DU-HL60′3, DU-HL60′4, TE5, and V1 were not expressed or were expressed poorly in MPs; they were expressed normally in control and treated UMs. However, HLA-DR expression was increased in treated UMs and MPs. The binding of the lectins RCA, Con A, WGA, DBA, UEA, and PNA was equivalent in all cells. Thus, MPs formed by fusion of UMs in vitro after culture with IFN-gamma differ in several features from UMs.

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

1994 ◽  
Vol 14 (2) ◽  
pp. 1364-1373
Author(s):  
A Eilers ◽  
M Baccarini ◽  
F Horn ◽  
R A Hipskind ◽  
C Schindler ◽  
...  
Keyword(s):  
Growth Factor ◽  
Dna Binding ◽  
Binding Protein ◽  
Trna Synthetase ◽  
Gamma Interferon ◽  
U937 Cells ◽  
Gamma Activation ◽  
Ifn Gamma ◽  
Differentiation Factors ◽  
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.

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

1993 ◽  
Vol 13 (6) ◽  
pp. 3245-3254 ◽  
Author(s):  
A Eilers ◽  
D Seegert ◽  
C Schindler ◽  
M Baccarini ◽  
T Decker
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Cellular Response ◽  
Gamma Interferon ◽  
U937 Cells ◽  
Monocytic Differentiation ◽  
Ifn Gamma ◽  
Exonuclease Iii ◽  
Activation Factor ◽  
Gel Shift

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.

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