Characterization of a tumor necrosis factor-responsive element which down-regulates the human osteocalcin gene

Tumor necrosis factor (TNF) down-regulates the production of bone matrix proteins by osteoblasts, thereby inhibiting bone formation. Osteocalcin, the major noncollagenous protein in bone, is inhibited by TNF at the transcriptional level. Mapping studies were undertaken to characterize the TNF-responsive element (TNFRE) in the osteocalcin promoter. Deletion analysis localized the TNFRE to the -522/-511 region, which contains a 9-bp palindromic motif (AGGCTGCCT). Promoter segments containing this sequence down-regulated a heterologous simian virus 40 promoter. Site-specific mutagenesis of the TNFRE eliminated TNF down-regulation. Mobility shift assays demonstrated that a constitutively expressed nuclear factor bound to the TNFRE; this factor was tentatively identified as the p50 homodimer of NF-kappa B. TNF stimulation induced a second TNFRE-binding protein which displaced the constitutive factor. The TNF-induced protein was not inhibitable by the NF-kappa B consensus sequence and was unreactive with anti-NF-kappa B antiserum. DNase footprinting demonstrated that both factors protected the -522/-501 portion of the promoter, consistent with the results of mapping studies and competitive mobility shift assays. It is hypothesized that the generalized catabolic activities of TNF in infectious and malignant diseases may be regulated via this novel element.

Download Full-text

Interleukin-6 induction by tumor necrosis factor and interleukin-1 in human fibroblasts involves activation of a nuclear factor binding to a kappa B-like sequence.

Molecular and Cellular Biology ◽

10.1128/mcb.10.7.3818 ◽

1990 ◽

Vol 10 (7) ◽

pp. 3818-3823 ◽

Cited By ~ 206

Author(s):

Y H Zhang ◽

J X Lin ◽

J Vilcek

Keyword(s):

Tumor Necrosis Factor ◽

Interleukin 6 ◽

Tumor Necrosis ◽

Interleukin 1 ◽

Human Fibroblasts ◽

Nf Kappa B ◽

Mobility Shift ◽

Dnase I Footprinting ◽

Chloramphenicol Acetyltransferase Gene ◽

Necrosis Factor

Using variable-length deletion constructs of the 5'-flanking region of the human interleukin-6 (IL-6) gene linked to the chloramphenicol acetyltransferase gene, we showed that the region from positions -109 to -50 mediated the bulk of the response to tumor necrosis factor (TNF) or interleukin-1 (IL-1), while it was less responsive to forskolin. DNA mobility shift assays and DNase I footprinting analysis identified a nuclear protein from TNF- or IL-1-treated fibroblasts that bound to a region comprising a kappa B-like element located between positions -72 and -63 on the IL-6 gene. On the basis of these and other experiments, we conclude that TNF and IL-1 apparently activate IL-6 gene expression by closely related mechanisms involving activation of a NF-kappa B-like factor, whereas the pathway of IL-6 induction by forskolin is, in part, different.

Download Full-text

Interleukin-6 induction by tumor necrosis factor and interleukin-1 in human fibroblasts involves activation of a nuclear factor binding to a kappa B-like sequence

Molecular and Cellular Biology ◽

10.1128/mcb.10.7.3818-3823.1990 ◽

1990 ◽

Vol 10 (7) ◽

pp. 3818-3823

Author(s):

Y H Zhang ◽

J X Lin ◽

J Vilcek

Keyword(s):

Tumor Necrosis Factor ◽

Interleukin 6 ◽

Tumor Necrosis ◽

Interleukin 1 ◽

Human Fibroblasts ◽

Nf Kappa B ◽

Mobility Shift ◽

Dnase I Footprinting ◽

Chloramphenicol Acetyltransferase Gene ◽

Necrosis Factor

Using variable-length deletion constructs of the 5'-flanking region of the human interleukin-6 (IL-6) gene linked to the chloramphenicol acetyltransferase gene, we showed that the region from positions -109 to -50 mediated the bulk of the response to tumor necrosis factor (TNF) or interleukin-1 (IL-1), while it was less responsive to forskolin. DNA mobility shift assays and DNase I footprinting analysis identified a nuclear protein from TNF- or IL-1-treated fibroblasts that bound to a region comprising a kappa B-like element located between positions -72 and -63 on the IL-6 gene. On the basis of these and other experiments, we conclude that TNF and IL-1 apparently activate IL-6 gene expression by closely related mechanisms involving activation of a NF-kappa B-like factor, whereas the pathway of IL-6 induction by forskolin is, in part, different.

Download Full-text

Fish oil enhances macrophage tumor necrosis factor-alpha mRNA expression at the transcriptional level

Metabolism ◽

10.1016/0026-0495(95)90196-5 ◽

1995 ◽

Vol 44 (6) ◽

pp. 800-805 ◽

Cited By ~ 18

Author(s):

Hernan R. Chang ◽

Denis Arsenijevic ◽

Ion-Rusan Vladoianu ◽

Lucien Girardier ◽

Abdul G. Dulloo

Keyword(s):

Tumor Necrosis Factor ◽

Fish Oil ◽

Mrna Expression ◽

Tumor Necrosis Factor Alpha ◽

Tumor Necrosis ◽

Transcriptional Level ◽

Necrosis Factor Alpha ◽

Factor Alpha ◽

Necrosis Factor

Download Full-text

The tumor suppressor p53 regulates its own transcription

Molecular and Cellular Biology ◽

10.1128/mcb.13.6.3415-3423.1993 ◽

1993 ◽

Vol 13 (6) ◽

pp. 3415-3423

Author(s):

A Deffie ◽

H Wu ◽

V Reinke ◽

G Lozano

Keyword(s):

Consensus Sequence ◽

Point Mutations ◽

Direct Interaction ◽

Mutant P53 ◽

Tumor Suppressor P53 ◽

Mobility Shift ◽

Shift Analysis ◽

Promoter Deletion Analysis ◽

Protein Dna Interactions ◽

Responsive Element

The ability of p53 to suppress transformation correlates with its ability to activate transcription. To identify targets of p53 transactivation, we examined the p53 promoter itself. Northern (RNA) analysis and transient transfection experiments showed that p53 transcriptionally regulated itself. A functionally inactive mutant p53 could not regulate the p53 promoter. Deletion analysis of the p53 promoter delineated sequences between +22 and +67 as being critical for regulation. Electrophoretic mobility shift analysis and methylation interference pinpointed the p53 DNA responsive element. When oligomerized in front of a heterologous minimal promoter, this element was regulated by wild-type p53 and not by mutant p53. Point mutations in the DNA element that eliminated protein-DNA interactions also resulted in a nonresponsive p53 promoter. The DNA element in the p53 promoter responsive to p53 regulation is similar to the p53 consensus sequence. However, we have been unable to detect a direct interaction of p53 with its promoter.

Download Full-text

Induction of the gene encoding mucosal vascular addressin cell adhesion molecule 1 by tumor necrosis factor alpha is mediated by NF-kappa B proteins.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.92.8.3561 ◽

1995 ◽

Vol 92 (8) ◽

pp. 3561-3565 ◽

Cited By ~ 53

Author(s):

M. Takeuchi ◽

V. R. Baichwal

Keyword(s):

Cell Adhesion ◽

Tumor Necrosis Factor ◽

Tumor Necrosis ◽

Cell Adhesion Molecule ◽

Nf Kappa B ◽

Gene Encoding ◽

Necrosis Factor Alpha ◽

Factor Alpha ◽

Cell Adhesion Molecule 1 ◽

Necrosis Factor

Download Full-text

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

Molecular and Cellular Biology ◽

10.1128/mcb.13.11.7191 ◽

1993 ◽

Vol 13 (11) ◽

pp. 7191-7198 ◽

Cited By ~ 289

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.

Download Full-text

Cyclic AMP-independent activation of transcription factor NF-kappa B in HL60 cells by tumor necrosis factors alpha and beta.

Molecular and Cellular Biology ◽

10.1128/mcb.11.4.2315 ◽

1991 ◽

Vol 11 (4) ◽

pp. 2315-2318 ◽

Cited By ~ 16

Author(s):

H P Hohmann ◽

R Kolbeck ◽

R Remy ◽

A P van Loon

Keyword(s):

Transcription Factor ◽

Tumor Necrosis Factor ◽

Cyclic Amp ◽

Tumor Necrosis ◽

Tnf Alpha ◽

Nf Kappa B ◽

Tumor Necrosis Factors ◽

Hl60 Cells ◽

Activation Of Transcription ◽

Necrosis Factor

No correlation exists in HL60 cells between NF-kappa B activation by tumor necrosis factor (TNF alpha) and TNF beta and intracellular levels of cyclic AMP. Cyclic AMP levels did not increase upon treatment of cells with each of these cytokines, although NF-kappa B was activated. Forskolin or 1-isobutyl-3-methylxanthine drastically increased intracellular levels of cyclic AMP, but neither activated NF-kappa B nor influenced TNF-induced NF-kappa B activation.

Download Full-text

Tumor necrosis factor and interleukin-1 lead to phosphorylation and loss of I kappa B alpha: a mechanism for NF-kappa B activation.

Molecular and Cellular Biology ◽

10.1128/mcb.13.6.3301 ◽

1993 ◽

Vol 13 (6) ◽

pp. 3301-3310 ◽

Cited By ~ 687

Author(s):

A A Beg ◽

T S Finco ◽

P V Nantermet ◽

A S Baldwin

Keyword(s):

Tumor Necrosis Factor ◽

Tumor Necrosis ◽

Interleukin 1 ◽

Activation Process ◽

Nf Kappa B ◽

I Kappa B Alpha ◽

Subsequent Loss ◽

Autoregulatory Mechanism ◽

Necrosis Factor

Nuclear factor kappa B (NF-kappa B) is a critical regulator of several genes which are involved in immune and inflammation responses. NF-kappa B, consisting of a 50-kDa protein (p50) and a 65-kDa protein (p65), is bound to a cytoplasmic retention protein called I kappa B. Stimulation of cells with a variety of inducers, including cytokines such as tumor necrosis factor and interleukin-1, leads to the activation and the translocation of p50/65 NF-kappa B into the nucleus. However, the in vivo mechanism of the activation process remains unknown. Here, we provide the first evidence that the in vivo mechanism of NF-kappa B activation is through the phosphorylation and subsequent loss of its inhibitor, I kappa B alpha. We also show that both I kappa B alpha loss and NF-kappa B activation are inhibited in the presence of antioxidants, demonstrating that the loss of I kappa B alpha is a prerequisite for NF-kappa B activation. Finally, we demonstrate that I kappa B alpha is rapidly resynthesized after loss, indicating that an autoregulatory mechanism is involved in the regulation of NF-kappa B function. We propose a mechanism for the activation of NF-kappa B through the modification and loss of I kappa B alpha, thereby establishing its role as a mediator of NF-kappa B activation.

Download Full-text