Transcriptional regulation of the human monocyte chemoattractant protein-1 gene: Cooperation of two NF-κB sites and NF-κB/Rel subunit specificity

Atsuhisa Ueda, Yoshiaki Ishigatsubo, Takao Okubo, Teizo Yoshimura

Research output: Contribution to journalArticle

267 Citations (Scopus)

Abstract

Human monocyte chemoattractant protein-1 (human MCP-1) mRNA accumulated in THP-1 cells 2 h after lipopolysaccharide (LPS) stimulation. DNase I footprinting revealed that LPS stimulation induced protein binding to the two closely located NF-κB sites, A1 and A2. By electrophoretic gel mobility shift assay and supershift assay, the binding of (p65)2, c-Rel/p65, p50/p65, and p50/c-Rel to the A2 oligonucleotide probe was detected after LPS stimulation. In contrast, 12-o-tetradecanoylphorbol 13-acetate did not induce a significant amount of MCP-1 mRNA in THP-1 cells 2 h after stimulation, and only p50/p65 bound to the A2 probe, trans-Activity of each NF-κB/Rel dimer was investigated by transfecting P19 cells with p65, p50, and/or c-Rel expression vectors, and a luciferase construct containing the enhancer region of the human MCP-1 gene. Expression of recombinant p65 or p65 and c-Rel resulted in elevated luciferase activities, indicating that (p65)2 and c- Rel/p65 had trans-activity. The binding of (p65)2 and/or c-Rel/p65 to the A2 probe was also detected from 12-o-tetradecanoylphorbol 13-acetate-stimulated HeLa, HOS, and A172 cells in which expression of MCP-1 mRNA was elevated. Finally, the role of the A1 site was investigated. Both (p65)2 and c- Rel/p65 bound to the A1 probe by electrophoretic mobility shift assay and a mutation in the A1 or A2 site resulted in a less of the enhancer activity. These results suggest that the binding of (p65)2 and c-Rel/p65 to the A1 and A2 sites of this gene is important for the tissue- and stimulus-specific transcription of the human MCP-1 gene.

Original languageEnglish
Pages (from-to)31092-31099
Number of pages8
JournalJournal of Biological Chemistry
Volume272
Issue number49
DOIs
Publication statusPublished - Dec 5 1997
Externally publishedYes

Fingerprint

varespladib methyl
Lipopolysaccharides
Assays
Genes
Luciferases
Messenger RNA
Acetates
Electrophoretic mobility
Oligonucleotide Probes
Deoxyribonuclease I
Electrophoretic Mobility Shift Assay
Tetradecanoylphorbol Acetate
Transcription
Dimers
Gels
Tissue
Protein Binding
human CCL2 protein
Mutation

ASJC Scopus subject areas

  • Biochemistry

Cite this

Transcriptional regulation of the human monocyte chemoattractant protein-1 gene : Cooperation of two NF-κB sites and NF-κB/Rel subunit specificity. / Ueda, Atsuhisa; Ishigatsubo, Yoshiaki; Okubo, Takao; Yoshimura, Teizo.

In: Journal of Biological Chemistry, Vol. 272, No. 49, 05.12.1997, p. 31092-31099.

Research output: Contribution to journalArticle

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