NF-κB Activation Is Required for C5a-Induced Interleukin-8 Gene Expression in Mononuclear Cells

Blood ◽  
1999 ◽  
Vol 93 (10) ◽  
pp. 3241-3249 ◽  
Author(s):  
Matthew H. Hsu ◽  
Meiying Wang ◽  
Darren D. Browning ◽  
Naofumi Mukaida ◽  
Richard D. Ye
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Pertussis Toxin ◽  
Mononuclear Cells ◽  
Binding Activity ◽  
Dominant Negative ◽  
Interleukin 8 ◽  
Peripheral Blood Mononuclear ◽  
C5a Receptor ◽  
Dna Binding Activity

C5a, a potent peptide chemoattractant, stimulates interleukin-8 (IL-8) secretion from peripheral blood mononuclear cells (PBMC). Experiments were conducted to understand the mechanisms for C5a-induced IL-8 production, which was 14-fold greater than that in unstimulated cells by 2 hours. IL-8 secretion was accompanied by accumulation of IL-8 mRNA in the cytosol and by nuclear expression of a κB DNA binding activity within 30 minutes. AP-1 but not NF-IL-6 DNA binding activity was also detected in C5a-stimulated PBMC; however, its delayed expression (maximal at 4 hours) suggested a less important role in the rapid production of IL-8. The correlation between C5a-induced κB binding activity and IL-8 gene expression was examined in the RAW264.7 macrophage cells using reporter genes directed by the κB sequence from IκB and IL-8 promoter regions. C5a-induced reporter gene expression was abolished by introducing mutations into the κB sites and by coexpression of a dominant negative IκB construct resistant to agonist-induced phosphorylation. Pertussis toxin, which ADP-ribosylates the Gi proteins known to couple to the C5a receptor, produced minimal inhibition of C5a-induced IL-8 expression and had little effect on C5a-induced calcium mobilization in RAW264.7 cells. These results suggest that NF-κB activation is required for C5a-induced IL-8 gene expression and that this response is mediated primarily through a pertussis toxin-insensitive pathway.

Extinction of insulin gene expression in hybrids between beta cells and fibroblasts is accompanied by loss of the putative beta-cell-specific transcription factor IEF1

1991 ◽  
Vol 11 (3) ◽  
pp. 1547-1552
Author(s):  
D Leshkowitz ◽  
M D Walker
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Binding Activity ◽  
Insulin Gene ◽  
Hybrid Cells ◽  
Dna Binding Activity ◽  
Insulin Producing Cells ◽  
Insulin Gene Expression ◽  
Insulinoma Cells

Insulin-producing cells and fibroblasts were fused to produce hybrid lines. In hybrids derived from both hamster and rat insulinoma cells, no insulin mRNA could be detected in any of seven lines examined by Northern (RNA) analysis despite the presence in each line of the insulin genes of both parental cells. Hybrid cells were transfected with recombinant chloramphenicol acetyltransferase plasmids containing defined segments of the rat insulin I gene 5' flank. We observed no transcriptional activity of the intact insulin enhancer or of IEB2, a critical cis-acting element of the insulin enhancer. IEB2 has previously been shown to interact in vitro with IEF1, a DNA-binding activity observed selectively in insulin-producing cells. Hybrid cells showed no detectable IEF1 activity. Furthermore, the insulin enhancer was unable to reduce transcription directed by the Moloney sarcoma virus enhancer in a double-enhancer construct. Thus, extinction of insulin gene expression in the hybrids apparently does not operate through a direct action of repressors on the insulin enhancer; rather, extinction is accompanied by, and may be caused by, reduced DNA-binding activity of the putative transcriptional activator IEF1.

Characterization of a dominant negativeC. elegansTwist mutant protein with implications for human Saethre-Chotzen syndrome

Development ◽  
2002 ◽  
Vol 129 (11) ◽  
pp. 2761-2772
Author(s):  
Ann K. Corsi ◽  
Thomas M. Brodigan ◽  
Erik M. Jorgensen ◽  
Michael Krause
Keyword(s):  
Dna Binding ◽  
Binding Activity ◽  
Binding Domain ◽  
Dominant Negative ◽  
Mutant Protein ◽  
Dna Binding Domain ◽  
Mesodermal Cell ◽  
C Elegans ◽  
Dna Binding Activity ◽  
A Charge

Twist is a transcription factor that is required for mesodermal cell fates in all animals studied to date. Mutations of this locus in humans have been identified as the cause of the craniofacial disorder Saethre-Chotzen syndrome. The Caenorhabditis elegans Twist homolog is required for the development of a subset of the mesoderm. A semidominant allele of the gene that codes for CeTwist, hlh-8, has defects that occur earlier in the mesodermal lineage than a previously studied null allele of the gene. The semidominant allele has a charge change (E29K) in the basic DNA-binding domain of CeTwist. Surprisingly, the mutant protein retains DNA-binding activity as both a homodimer and a heterodimer with its partner E/Daughterless (CeE/DA). However, the mutant protein blocks the activation of the promoter of a target gene. Therefore, the mutant CeTwist may cause cellular defects as a dominant negative protein by binding to target promoters as a homo- or heterodimer and then blocking transcription. Similar phenotypes as those caused by the E29K mutation were observed when amino acid substitutions in the DNA-binding domain that are associated with the human Saethre-Chotzen syndrome were engineered into the C. elegans protein. These data suggest that Saethre-Chotzen syndrome may be caused, in some cases, by dominant negative proteins, rather than by haploinsufficiency of the locus.

scholarly journals Overexpression of mitogen-activated protein kinase (ERK1) enhances T- cell cytokine gene expression: role of AP1, NF-AT, and NF-KB

Blood ◽  
1993 ◽  
Vol 82 (8) ◽  
pp. 2470-2477 ◽  
Author(s):  
JH Park ◽  
L Levitt
Keyword(s):  
Gene Expression ◽  
T Cell ◽  
Dna Binding ◽  
Map Kinase ◽  
Binding Activity ◽  
Cytokine Gene Expression ◽  
Cytokine Gene ◽  
Dna Binding Activity ◽  
Mitogen Activated Protein

Abstract Transfected Jurkat cells overexpressing extracellular signal-regulated kinase (ERK1), also referred to as mitogen-activated protein (MAP) kinase, were selected by Western blotting assay using anti-ERK1 and antiphosphotyrosine antibodies in combination with a functional MAP kinase assay. We then asked whether enhanced ERK1 expression had any effect on induction of T-cell cytokine genes. The results show that overexpression of ERK1 enhances expression of T-cell interleukin-2 (IL- 2), IL-3, and granulocyte-macrophage colony-stimulating factor mRNA; no change was seen in expression of the alpha-actin gene. DNA-binding activities of the transcription factors AP1, NF-AT, and NF-kB were specifically increased twofold to fourfold in ERK1-overexpressing clones relative to nontransformed or vector-transformed cells, whereas no enhancement of CK1-CK2 protein DNA binding activity was detected after ERK1 overexpression. Additionally, increased NF-AT DNA binding activity was associated with functional enhancement of NF-AT transactivating activity in ERK1-overexpressing cells. These results provide direct evidence for the role of MAP kinase in the regulation of cytokine gene expression and indicate that such regulation is likely mediated through the enhanced DNA binding activity of specific nuclear transcription factors.

Treatment of cultured myotubes with the proteasome inhibitor β-lactone increases the expression of the transcription factor C/EBPβ

2007 ◽  
Vol 292 (1) ◽  
pp. C216-C226 ◽  
Author(s):  
Wei Wei ◽  
Hongmei Yang ◽  
Michael Menconi ◽  
Peirang Cao ◽  
Chester E. Chamberlain ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Transcription Factor ◽  
Dna Binding ◽  
Proteasome Inhibitor ◽  
Binding Activity ◽  
Dominant Negative ◽  
Dna Binding Activity ◽  
Cultured Myotubes ◽  
Nuclear Levels ◽  
Factor C

The role of the proteasome in the regulation of cellular levels of the transcription factor CCAAT/enhancer-binding protein β (C/EBPβ) is poorly understood. We tested the hypothesis that C/EBPβ levels in cultured myotubes are regulated, at least in part, by proteasome activity. Treatment of cultured L6 myotubes, a rat skeletal muscle cell line, with the specific proteasome inhibitor β-lactone resulted in increased nuclear levels of C/EBPβ as determined by Western blotting and immunofluorescent detection. This effect of β-lactone reflected inhibited degradation of C/EBPβ. Surprisingly, the increased C/EBPβ levels in β-lactone-treated myotubes did not result in increased DNA-binding activity. In additional experiments, treatment of the myotubes with β-lactone resulted in increased nuclear levels of growth arrest DNA damage/C/EBP homologous protein (Gadd153/CHOP), a dominant-negative member of the C/EBP family that can form heterodimers with other members of the C/EBP family and block DNA binding. Coimmunoprecipitation and immunofluorescent detection provided evidence that C/EBPβ and Gadd153/CHOP interacted and colocalized in the nuclei of the β-lactone-treated myotubes. When Gadd153/CHOP expression was downregulated by transfection of myotubes with siRNA targeting Gadd153/CHOP, C/EBPβ DNA-binding activity was restored in β-lactone-treated myotubes. The results suggest that C/EBPβ is degraded by a proteasome-dependent mechanism in skeletal muscle cells and that Gadd153/CHOP can interact with C/EBPβ and block its DNA-binding activity. The observations are important because they increase the understanding of the complex regulation of the expression and activity of C/EBPβ in skeletal muscle.

scholarly journals Recruitment of the tinman homolog Nkx-2.5 by serum response factor activates cardiac alpha-actin gene transcription.

1996 ◽  
Vol 16 (11) ◽  
pp. 6372-6384 ◽  
Author(s):  
C Y Chen ◽  
R J Schwartz
Keyword(s):  
Dna Binding ◽  
Serum Response Factor ◽  
Binding Activity ◽  
Dominant Negative ◽  
Actin Gene ◽  
Response Factor ◽  
Dna Binding Activity ◽  
Actin Promoter ◽  
Alpha Actin ◽  
Serum Response

We recently showed that the cardiogenic homeodomain factor Nkx-2.5 served as a positive acting accessory factor for serum response factor (SRF) and that together they provided strong transcriptional activation of the cardiac alpha-actin promoter, depending upon intact serum response elements (SREs) (C. Y. Chen, J. Croissant, M. Majesky, S. Topouz, T. McQuinn, M. J. Frankovsky, and R. J. Schwartz, Dev. Genet. 19:119-130, 1996). As shown here, Nkx-2.5 and SRF collaborated to activate the endogenous murine cardiac alpha-actin gene in 10T1/2 fibroblasts by a mechanism in which SRF recruited Nkx-2.5 to the alpha-actin promoter. Activation of a truncated promoter consisting of the proximal alpha-actin SRE1 occurred even when Nkx-2.5 DNA-binding activity was blocked by a point mutation in the third helix of its homeodomain. Investigation of protein-protein interactions showed that Nkx-2.5 was bound to SRF in the absence of DNA in soluble protein complexes retrieved from cardiac myocyte nuclei but could also be detected in coassociated binding complexes on the proximal SRE1. Recruitment of Nkx-2.5 to an SRE depended upon SRF DNA-binding activity and was blocked by the dominant negative SRFpm1 mutant, which allowed for dimerization of SRF monomers but prevented DNA binding. Interactive regions shared by Nkx-2.5 and SRF were mapped to N-terminal/helix I and helix II/helix III regions of the Nkx-2.5 homeodomain and to the N-terminal extension of the MADS box. Our study suggests that physical association between Nkx-2.5 and SRF is one way that cardiac specified genes are activated in cardiac cell lineages.

CREB trans-activates the murine H+-K+-ATPase α2-subunit gene

2004 ◽  
Vol 287 (4) ◽  
pp. C903-C911 ◽  
Author(s):  
Xiangyang Xu ◽  
Wenzheng Zhang ◽  
Bruce C. Kone
Keyword(s):  
Dna Binding ◽  
Transcriptional Control ◽  
Luciferase Activity ◽  
Collecting Duct ◽  
Binding Activity ◽  
Dominant Negative ◽  
Proximal Promoter ◽  
Mrna Levels ◽  
Dna Binding Activity

Despite its key role in potassium homeostasis, transcriptional control of the H+-K+-ATPase α2-subunit (HKα2) gene in the collecting duct remains poorly characterized. cAMP increases H+-K+-ATPase activity in the collecting duct, but its role in activating HKα2 transcription has not been explored. Previously, we demonstrated that the proximal 177 bp of the HKα2 promoter confers basal collecting duct-selective expression. This region contains several potential cAMP/Ca2+-responsive elements (CRE). Accordingly, we examined the participation of CRE-binding protein (CREB) in HKα2 transcriptional control in murine inner medullary collecting duct (mIMCD)-3 cells. Forskolin and vasopressin induced HKα2 mRNA levels, and CREB overexpression stimulated the activity of HKα2 promoter-luciferase constructs. Serial deletion analysis revealed that CREB inducibility was retained in a construct containing the proximal 100 bp of the HKα2 promoter. In contrast, expression of a dominant negative inhibitor (A-CREB) resulted in 60% lower HKα2 promoter-luciferase activity, suggesting that constitutive CREB participates in basal HKα2 transcriptional activity. A constitutively active CREB mutant (CREB-VP16) strongly induced HKα2 promoter-luciferase activity, whereas overexpression of CREBdLZ-VP16, which lacks the CREB DNA-binding domain, abolished this activation. In vitro DNase I footprinting and gel shift/supershift analysis of the proximal promoter with recombinant glutathione S-transferase (GST)-CREB-1 and mIMCD-3 cell nuclear extracts revealed sequence-specific DNA-CREB-1 complexes at −86/−60. Mutation at three CRE-like sequences within this region abolished CREB-1 DNA-binding activity and abrogated CREB-VP16 trans-activation of the HKα2 promoter. In contrast, mutation of the neighboring −104/−94 κβ element did not alter CREB-VP16 trans-activation of the HKα2 promoter. Thus CREB-1, binding to one or more CRE-like elements in the −86/−60 region, trans-activates the HKα2 gene and may represent an important link between rapid and delayed effects of cAMP on HKα2 activity.

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