Transcriptional regulation of the human monocyte chemoattractant protein-1 gene -: Cooperation of two NF-κB sites and NF-κB/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-kappa B 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-kappa B/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 Al site was investigated, Both (p65)(2) and c-Rel/p65 bound to the Al 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.