Transcriptional regulation of the human monocyte chemoattractant protein-1 gene. Cooperation of two NF-kappaB sites and NF-kappaB/Rel subunit specificity.
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-kappaB 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-kappaB/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 loss 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.
Ueda
A
A
Immunopathology Section, Laboratory of Immunobiology, National Cancer Institute-Frederick, Cancer Research and Development Center, Frederick, Maryland 21702, USA.
Ishigatsubo
Y
Y
Okubo
T
T
Yoshimura
T
T
D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D018932:Chemokine CCL2 / Q000235:genetics*; D004247:DNA / Q000378:metabolism; D018983:DNA Footprinting; D019281:Dimerization; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism / Q000502:physiology*; D052002:NF-kappa B p50 Subunit; D011485:Protein Binding; D011487:Protein Conformation; D012333:RNA, Messenger / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D051996:Transcription Factor RelA; D014158:Transcription, Genetic
D018932:Chemokine CCL2; D008070:Lipopolysaccharides; D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; D012333:RNA, Messenger; D051996:Transcription Factor RelA; D004247:DNA; D013755:Tetradecanoylphorbol Acetate