scholarly journals Transcriptional regulation of NF-kappa B2: evidence for kappa B-mediated positive and negative autoregulation.

1994 ◽  
Vol 14 (12) ◽  
pp. 7695-7703 ◽  
Author(s):  
S Liptay ◽  
R M Schmid ◽  
E G Nabel ◽  
G J Nabel
Keyword(s):  
Transcriptional Regulation ◽  
Transcriptional Repression ◽  
Genomic Structure ◽  
Family Members ◽  
Regulatory Elements ◽  
Binding Activity ◽  
Nf Kappa B ◽  
Necrosis Factor Alpha ◽  
Repressive Effect ◽  
Factor Alpha

NF-kappa B is an inducible transcription factor complex which regulates the expression of a variety of genes which are involved in the immune, inflammatory, and acute-phase responses. The maintenance of NF-kappa B activity in stimulated cells requires ongoing protein synthesis, suggesting several modes of regulation. In this report, we have characterized the transcriptional regulation of one family member, NF-kappa B2. The genomic structure and sequence of NF-kappa B2 revealed the presence of two promoters and at least four kappa B regulatory elements, which mediate responsiveness to phorbol myristate acetate and tumor necrosis factor alpha. Similar to other NF-kappa B family members, NF-kappa B2 is positively autoregulated. In contrast to other family members, we find that kappa B elements in the NFKB2 promoter can also mediate transcriptional repression in the absence of NF-kappa B. We identified a nuclear complex which binds specifically to a subset of kappa B-related sites but not to the canonical kappa B element. Because of its putative inhibitory or repressive effect, this binding activity has been termed Rep-kappa B. This mechanism of repressing basal NF-kappa B2 transcription in an inactivated state enables the cell to tightly control NF-kappa B2 activity. These data demonstrate that a novel mode of kappa B-dependent regulation is mediated by specific kappa B sites in the NFKB2 promoter.

Transcriptional regulation of NF-kappa B2: evidence for kappa B-mediated positive and negative autoregulation

1994 ◽  
Vol 14 (12) ◽  
pp. 7695-7703
Author(s):  
S Liptay ◽  
R M Schmid ◽  
E G Nabel ◽  
G J Nabel
Keyword(s):  
Transcriptional Regulation ◽  
Transcriptional Repression ◽  
Genomic Structure ◽  
Family Members ◽  
Regulatory Elements ◽  
Binding Activity ◽  
Nf Kappa B ◽  
Necrosis Factor Alpha ◽  
Repressive Effect ◽  
Factor Alpha

NF-kappa B is an inducible transcription factor complex which regulates the expression of a variety of genes which are involved in the immune, inflammatory, and acute-phase responses. The maintenance of NF-kappa B activity in stimulated cells requires ongoing protein synthesis, suggesting several modes of regulation. In this report, we have characterized the transcriptional regulation of one family member, NF-kappa B2. The genomic structure and sequence of NF-kappa B2 revealed the presence of two promoters and at least four kappa B regulatory elements, which mediate responsiveness to phorbol myristate acetate and tumor necrosis factor alpha. Similar to other NF-kappa B family members, NF-kappa B2 is positively autoregulated. In contrast to other family members, we find that kappa B elements in the NFKB2 promoter can also mediate transcriptional repression in the absence of NF-kappa B. We identified a nuclear complex which binds specifically to a subset of kappa B-related sites but not to the canonical kappa B element. Because of its putative inhibitory or repressive effect, this binding activity has been termed Rep-kappa B. This mechanism of repressing basal NF-kappa B2 transcription in an inactivated state enables the cell to tightly control NF-kappa B2 activity. These data demonstrate that a novel mode of kappa B-dependent regulation is mediated by specific kappa B sites in the NFKB2 promoter.

scholarly journals Three NF-kappa B binding sites in the human E-selectin gene required for maximal tumor necrosis factor alpha-induced expression.

1994 ◽  
Vol 14 (9) ◽  
pp. 5820-5831 ◽  
Author(s):  
U Schindler ◽  
V R Baichwal
Keyword(s):  
Endothelial Cells ◽  
Tumor Necrosis Factor ◽  
Binding Sites ◽  
Tumor Necrosis ◽  
Regulatory Elements ◽  
Tnf Alpha ◽  
Nf Kappa B ◽  
Necrosis Factor Alpha ◽  
Factor Alpha ◽  
Necrosis Factor

Transcription of the gene encoding the endothelial cell-leukocyte adhesion molecule (ELAM-1; E-selectin) is induced in response to various cytokines, including tumor necrosis factor-alpha (TNF-alpha) and interleukin-1. A DNase I-hypersensitive site in the 5' proximal promoter region of the E-selectin gene is observed in human umbilical vein endothelial cells only following TNF-alpha treatment, suggesting the presence of a TNF-alpha-inducible element close to the transcriptional start site. Transient transfection studies in endothelial cells demonstrated that 170 bp of upstream sequences is sufficient to confer TNF-alpha inducibility. Systematic site-directed mutagenesis of this region revealed two regulatory elements (-129 to -110 and -99 to -80) that are essential for maximal promoter activity following cytokine treatment. Protein binding studies with crude nuclear extracts and recombinant proteins revealed that the two elements correspond to three NF-kappa B binding sites (site 1, -126; site 2, 116; and site 3, -94). All three sites can be bound by NF-kappa B when used as independent oligonucleotides in mobility shift assays. However, within the context of a larger promoter fragment, sites 2 and 3 are preferentially occupied over site 1. These data are consistent with results obtained in transfection studies demonstrating that mutations in sites 2 and 3 are more detrimental than mutations within site 1. Hence, inducibility of the E-selectin gene requires the interaction of NF-kappa B proteins bound to multiple regulatory elements.

Three NF-kappa B binding sites in the human E-selectin gene required for maximal tumor necrosis factor alpha-induced expression

1994 ◽  
Vol 14 (9) ◽  
pp. 5820-5831
Author(s):  
U Schindler ◽  
V R Baichwal
Keyword(s):  
Endothelial Cells ◽  
Tumor Necrosis Factor ◽  
Binding Sites ◽  
Tumor Necrosis ◽  
Regulatory Elements ◽  
Tnf Alpha ◽  
Nf Kappa B ◽  
Necrosis Factor Alpha ◽  
Factor Alpha ◽  
Necrosis Factor

Transcription of the gene encoding the endothelial cell-leukocyte adhesion molecule (ELAM-1; E-selectin) is induced in response to various cytokines, including tumor necrosis factor-alpha (TNF-alpha) and interleukin-1. A DNase I-hypersensitive site in the 5' proximal promoter region of the E-selectin gene is observed in human umbilical vein endothelial cells only following TNF-alpha treatment, suggesting the presence of a TNF-alpha-inducible element close to the transcriptional start site. Transient transfection studies in endothelial cells demonstrated that 170 bp of upstream sequences is sufficient to confer TNF-alpha inducibility. Systematic site-directed mutagenesis of this region revealed two regulatory elements (-129 to -110 and -99 to -80) that are essential for maximal promoter activity following cytokine treatment. Protein binding studies with crude nuclear extracts and recombinant proteins revealed that the two elements correspond to three NF-kappa B binding sites (site 1, -126; site 2, 116; and site 3, -94). All three sites can be bound by NF-kappa B when used as independent oligonucleotides in mobility shift assays. However, within the context of a larger promoter fragment, sites 2 and 3 are preferentially occupied over site 1. These data are consistent with results obtained in transfection studies demonstrating that mutations in sites 2 and 3 are more detrimental than mutations within site 1. Hence, inducibility of the E-selectin gene requires the interaction of NF-kappa B proteins bound to multiple regulatory elements.

scholarly journals Distinct mechanisms for regulation of the interleukin-8 gene involve synergism and cooperativity between C/EBP and NF-kappa B.

1993 ◽  
Vol 13 (11) ◽  
pp. 7191-7198 ◽  
Author(s):  
B Stein ◽  
A S Baldwin
Keyword(s):  
Gene Expression ◽  
Tumor Necrosis Factor ◽  
Binding Site ◽  
Tumor Necrosis ◽  
Interleukin 8 ◽  
Cooperative Binding ◽  
Nf Kappa B ◽  
Necrosis Factor Alpha ◽  
Factor Alpha ◽  
Necrosis Factor

The interleukin-8 promoter is transcriptionally activated by interleukin-1, tumor necrosis factor alpha, phorbol myristate acetate, or hepatitis B virus X protein through a sequence located between positions -91 and -71. This region contains an NF-kappa B-like and a C/EBP-like binding site. We show here that several members of the NF-kappa B family, including p65, p50, p52, and c-Rel, can bind to this region, confirming an authentic NF-kappa B binding site in the interleukin-8 promoter. Further, C/EBP binds only weakly to the interleukin-8 promoter site. Electrophoretic mobility shift assays with proteins overexpressed in COS cells and with nuclear extracts from tumor necrosis factor alpha-stimulated HeLa cells demonstrated a strong cooperative binding of C/EBP to its site when NF-kappa B is bound to its adjacent binding site. Transfection studies lead to a model that suggests a highly complex regulation of interleukin-8 gene expression at multiple levels: independent binding of C/EBP and NF-kappa B to their respective sites, cooperative binding of C/EBP and NF-kappa B to DNA, and positive synergistic activation through the C/EBP binding site and inhibition through the NF-kappa B binding site by combinations of C/EBP and NF-kappa B. Thus, the ultimate regulation of interleukin-8 gene expression depends on the ratio of cellular C/EBP and NF-kappa B.

scholarly journals Activation of the adhesive capacity of CR3 on neutrophils by endotoxin: dependence on lipopolysaccharide binding protein and CD14.

1991 ◽  
Vol 173 (5) ◽  
pp. 1281-1286 ◽  
Author(s):  
S D Wright ◽  
R A Ramos ◽  
A Hermanowski-Vosatka ◽  
P Rockwell ◽  
P A Detmers
Keyword(s):  
Binding Protein ◽  
Binding Activity ◽  
Central Feature ◽  
Colony Stimulating Factor ◽  
Necrosis Factor Alpha ◽  
Formyl Peptide ◽  
Factor Alpha ◽  
Macrophage Colony ◽  
Stimulating Factor

Tumor necrosis factor alpha, granulocyte colony-stimulating factor, granulocyte/macrophage colony-stimulating factor, and formyl peptide were each found to cause a twofold increase in expression of CD14 on the surface of polymorphonuclear leukocytes (PMN). Upregulation of CD14 was complete by 20 min and thus appeared to result from expression of preformed stores of protein. The CD14 on the surface of PMN was shown to serve two biological functions. It bound particles coated with complexes of lipopolysaccharide (LPS) and LPS binding protein (LBP). This binding activity was enhanced by agonists that upregulated CD14 expression and may serve in the clearance of Gram-negative bacteria opsonized with LBP. Interaction of CD14 with LPS in the presence of LBP or serum also caused a dramatic, transient increase in the adhesive activity of CR3 (CD11b/CD18) on PMN. Enhanced activity of CR3 and other members of the CD11/CD18 family underlies many of the known physiological responses of PMN to LPS and may be a central feature of the in vivo responses of PMN to endotoxin.

scholarly journals Distinct mechanisms for regulation of the interleukin-8 gene involve synergism and cooperativity between C/EBP and NF-kappa B

1993 ◽  
Vol 13 (11) ◽  
pp. 7191-7198 ◽  
Author(s):  
B Stein ◽  
A S Baldwin
Keyword(s):  
Gene Expression ◽  
Tumor Necrosis Factor ◽  
Binding Site ◽  
Tumor Necrosis ◽  
Interleukin 8 ◽  
Cooperative Binding ◽  
Nf Kappa B ◽  
Necrosis Factor Alpha ◽  
Factor Alpha ◽  
Necrosis Factor

The interleukin-8 promoter is transcriptionally activated by interleukin-1, tumor necrosis factor alpha, phorbol myristate acetate, or hepatitis B virus X protein through a sequence located between positions -91 and -71. This region contains an NF-kappa B-like and a C/EBP-like binding site. We show here that several members of the NF-kappa B family, including p65, p50, p52, and c-Rel, can bind to this region, confirming an authentic NF-kappa B binding site in the interleukin-8 promoter. Further, C/EBP binds only weakly to the interleukin-8 promoter site. Electrophoretic mobility shift assays with proteins overexpressed in COS cells and with nuclear extracts from tumor necrosis factor alpha-stimulated HeLa cells demonstrated a strong cooperative binding of C/EBP to its site when NF-kappa B is bound to its adjacent binding site. Transfection studies lead to a model that suggests a highly complex regulation of interleukin-8 gene expression at multiple levels: independent binding of C/EBP and NF-kappa B to their respective sites, cooperative binding of C/EBP and NF-kappa B to DNA, and positive synergistic activation through the C/EBP binding site and inhibition through the NF-kappa B binding site by combinations of C/EBP and NF-kappa B. Thus, the ultimate regulation of interleukin-8 gene expression depends on the ratio of cellular C/EBP and NF-kappa B.

scholarly journals Regulation of tumor necrosis factor alpha transcription in macrophages: involvement of four kappa B-like motifs and of constitutive and inducible forms of NF-kappa B.

1990 ◽  
Vol 10 (4) ◽  
pp. 1498-1506 ◽  
Author(s):  
M A Collart ◽  
P Baeuerle ◽  
P Vassalli
Keyword(s):  
Tumor Necrosis Factor ◽  
Tumor Necrosis Factor Alpha ◽  
Tumor Necrosis ◽  
Tnf Alpha ◽  
Nf Kappa B ◽  
Necrosis Factor Alpha ◽  
Protein Subunits ◽  
Factor Alpha ◽  
Necrosis Factor ◽  
Stimulation Of

This study characterizes the interaction of murine macrophage nuclear proteins with the tumor necrosis factor alpha (TNF-alpha) promoter. Gel retardation and methylation interference assays showed that stimulation of TNF-alpha gene transcription in peritoneal exudate macrophages was accompanied by induction of DNA-binding proteins that recognized with different affinities four elements related to the kappa B consensus motif and a Y-box motif. We suggest that the basal level of TNF-alpha expression in macrophages is due to the binding of a constitutive form of NF-kappa B, present at low levels in nuclei from resting thioglycolate exudate peritoneal macrophages, to some if not all of the kappa B motifs; we postulate that this constitutive form contains only the 50-kilodalton (kDa) DNA-binding protein subunits of NF-kappa B, not the 65-kDa protein subunits (P. Baeuerle and D. Baltimore, Genes Dev. 3:1689-1698, 1989). Agents such as glucocorticoids, which decrease TNF-alpha transcription, diminished the basal level of nuclear NF-kappa B. Stimulation of Stimulation of TNF-alpha transcription in macrophages by lipopolysaccharide, gamma interferon, or cycloheximide led to an increased content of nuclear NF-kappa B. This induced factor represents a different form of NF-kappa B, since it generated protein-DNA complexes of slower mobility; we propose that this induced form of NF-kappa B contains both the 50- and 65-kDa protein subunits, the latter ones being necessary to bind NF-kappa B to its cytoplasmic inhibitor in uninduced cells (Baeuerle and Baltimore, Genes Dev., 1989). In resting cells, this inducible form of NF-kappa B was indeed detectable in the cytosol after deoxycholate treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

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