Cis-acting DNA Elements of Mouse Granulocyte/Macrophage Colony-stimulating Factor Gene Responsive to Oxidized Low Density Lipoprotein

• Published in: The Journal of biological chemistry Vol. 274; no. 53; pp. 37665 - 37672
• Main Authors: Matsumura, T, Sakai, M, Matsuda, K, Furukawa, N, Kaneko, K, Shichiri, M
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 31.12.1999
• Subjects: Animals; Base Sequence; Cell Line; DNA - genetics; DNA Primers; Granulocyte-Macrophage Colony-Stimulating Factor - genetics; Humans; Lipoproteins, LDL - metabolism; Mice; Nuclear Proteins - metabolism; Promoter Regions, Genetic; Protein Binding; Protein Kinase C - metabolism; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger - genetics; Transcription, Genetic
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.274.53.37665

We previously demonstrated that the induction of granulocyte/macrophage colony-stimulating factor (GM-CSF) played an important role in oxidized low density lipoprotein (Ox-LDL)-induced macrophage growth as a growth priming factor. The present study was undertaken to elucidate the transcriptional regulation of the GM-CSF gene using Raw 264.7 cells, a mouse macrophage cell line. Transient transfection into Raw 264.7 cells of several 5′-flanking regions of GM-CSF gene-luciferase fusion plasmids revealed the presence of two positive regulatory sites in regions spanning from −97 to −59 and from −59 to −37 and one negative regulatory site from −120 to −97 in unstimulated cells. When cells were stimulated by Ox-LDL, there was one positive responsive site from −225 to −120 and one negative responsive site from −97 to −59, which contained the NF-κB binding site. Computer analysis revealed the presence of a putative AP-2 binding site from −169 to −160. Mutagenesis of a putative AP-2 binding site and tandem repeat of this site in plasmid resulted in a complete loss and increased responsiveness to Ox-LDL, respectively. Electrophoretic mobility shift assay showed that Ox-LDL increased the binding of certain nuclear protein(s) to a putative AP-2 binding site but decreased their binding to NF-κB binding site. Supershift assay showed that nuclear proteins bound to NF-κB binding site contained, at least, p50 and p65 but could not demonstrate nuclear protein(s) bound to a putative AP-2 binding site. Our results suggested that a putative AP-2 binding site from −169 to −160 was a positive responsive element to Ox-LDL and that the NF-κB binding site from −91 to −82 was a negative responsive element in Ox-LDL-induced GM-CSF transcription.